Hyaluronic acid derivative and drug containing the same

ABSTRACT

A hyaluronic acid derivative in which an anti-inflammatory drug is bound to hyaluronic acid through a covalent bond via a spacer having a biodegradable region, and a production process thereof.

TECHNICAL FIELD

The present invention relates to hyaluronic acid derivatives to which anon-steroidal anti-inflammatory drug or a disease-modifyinganti-rheumatic drug is introduced via a spacer which is biodegradable,and production methods thereof.

BACKGROUND ART

A sodium hyaluronate solution is used as a therapeutic agent forarthritis such as osteoarthritis of knee (OA) or rheumatoid arthritis ofknee (RA). The sodium hyaluronate solution is generally used asinjections by the direct administration to the affected knee joints, andshoulder joints, and frequently used for the purpose of improvingfunctional disorders and suppressing pain caused by the arthritis.

Non-steroidal anti-inflammatory drugs (hereinafter also referred to as“NSAIDs” or “NSAID”) and disease-modifying anti-rheumatic drugs(hereinafter also referred to as “DMARD”), which improve morbid statessuch as articular rheumatism, are also used as agents for suppressing oralleviating pains caused by such arthritis. In general, these NSAIDs areorally administered in many cases, and there are also frequent cases inwhich concomitant use of the injection of the above-described sodiumhyaluronate solution and oral administration of NSAIDs. In the case ofthe oral administration of these NSAIDs, there is a problem in that thegreater part of the NSAIDs are metabolized while circulating in theblood stream before they reach the affected site. To circumvent thisproblem, high dosage of NSAIDs are necessary to maintain effectiveconcentration in the blood in order to deriver NSAIDs to the affectedpart. However, such high dosage of NSAIDs by the oral administrationcauses serious gastrointestinal adverse effects.

In addition, immunotherapy agents (immunomodulators andimmunosuppressants) are used as DMARD for controlling immune abnormalityor the like which is considered to be a cause of inflammation.

On the other hand, Hyaluronic acid is a polysaccharide constituted by arepeating structure with a disaccharide unit of N-acetyl-D-glucosamineand D-glucuronic acid as the basic core structure, and it is known to behighly hydrophilic due to the carboxyl group and a number of hydroxylgroup in the disaccharide unit. As an example that hyaluronic acid hashydrophilic property, namely high hydration with water molecule,hyaluronic acid can hold water about 1,000-fold larger than its ownweight. However, when highly hydrophobic agents such as NSAIDs areintroduced into hyaluronic acid having such a high hydrophilic property,it is conventionally known that hydrophobic property of hyaluronic acidmolecule itself increases so that water-semi-insoluble gel or insolublematter are formed. Consequently, those water-semi-insoluble gel orinsoluble matter are not suitable for the injectable use. Furthermore,with the increase of the degree of substitution of medicament for thepurpose of longer sustained release, the insolubility is also increasedso that it takes a form inappropriate as injections.

As an example in which not only NSAIDs but also other medicaments wereintroduced into hyaluronic acid, there is a report on a conjugate inwhich a matrix metalloproteinase inhibitor (MMP inhibitor) as anarthritis treating agent and hyaluronic acid were bound to each othervia a spacer or not via the spacer (Patent Reference 1). However, as asuitable binding mode of the MMP inhibitor with hyaluronic acid,stronger covalent bond is exemplified in the report, and it suggeststhat a synergistic medicament effect of the action of the MMP inhibitorand the effect of hyaluronic acid can be expected on the assumption thatthe conjugate are not dissociated and degraded into the MMP inhibitorand hyaluronic acid in the administered region. In addition, itexemplifies carboxyl group as the binding region with hyaluronic acid,however, the degree of substitution of the medicament to the carboxylgroup is considerably low, or a treatment for keeping suitableembodiment (solution) as injections is not carried out.

As other examples, there is a case in which hyaluronic acid is activatedwith water-soluble carbodiimide, and nucleophilic reagents were allowedto react therewith (Patent Reference 2), but these medicaments were notNSAIDs, and the final dosage form was an insoluble film. In addition,there is a case in which various medicaments were introduced intohyaluronic acid using a halogenated di-lower alkylphosphinothioyl(Rpt-X) as the condensing agent (Patent Reference 3), but dosage formsof the prepared derivatives are not described, and a treatment forenabling them as solution in the preparation is not included in theprocess.

-   Patent Reference 1: WO 99/59603-   Patent Reference 2: JP-T-3-502704-   Patent Reference 3: JP-A-9-188705

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

A method in which the problematic point as gastrointestinal adverseeffects caused by the oral administration of NSAIDs is avoided by directinjection NSAIDs into the affected site can be considered Althoughtheoretically, but for example, when NSAIDs are directly injected intothe knee joint cavity, the period of time for continuing the effect ofNSAIDs is short due to quick absorption, so that such a method is notadopted. In addition, since NSAIDs themselves aim at alleviating orsuppressing pain, such a method does not become a basic remedy forarthritis.

Accordingly, the present invention aims at providing a pharmaceuticalagent which can greatly contribute to the alleviation or suppression ofpain accompanied by arthritis and basic remedy for arthritis, bypreparing a novel derivative in which one of NSAIDs or DMARD ischemically introduced into an arthritis treating agent, sodiumhyaluronate, and injecting this into the affected site, and providing apharmaceutical agent which shows its prolonged effect through thecontrolled release of NSAIDs or DMARD.

Means for Solving the Problems

Taking the above-described problems into consideration, the presentinventors have conducted intensive studies with the aim of developingNSAIDs-introduced hyaluronic acid derivatives and DMARD-introducedhyaluronic acid derivatives, which can be used as injections into theaffected site of arthritis patients, and also have high effects in notonly radically treating arthritis but also alleviating or suppressingpain and inflammation.

As a result, it was found that derivatives in which NSAIDs and DMARD areintroduced into hyaluronic acid via a spacer having a biodegradableregion are suitable for the above-described objects, and furtherpreferably that soluble NSAIDs-introduced hyaluronic acid derivativesand soluble DMARD-introduced hyaluronic acid derivatives, which can beused as injectable solutions in the form of infusions (injections), canbe obtained through the improvement of solubility by adding an alkalitreatment to the production process, thereby accomplishing the presentinvention.

That is, the present invention relates to the followings:

(1) A hyaluronic acid derivatives in which anti-inflammatory drugs arebound to hyaluronic acid through a covalent bond via a spacer having abiodegradable region.(2) The hyaluronic acid derivatives according to the above-described(1), wherein the anti-inflammatory drugs are selected from non-steroidalanti-inflammatory drugs and disease-modifying anti-rheumatic drugs.(3) The hyaluronic acid derivatives according to the above-described (1)or (2), wherein the anti-inflammatory drugs have a carboxyl group.(4) The hyaluronic acid derivatives according to the above-described(3), wherein the anti-inflammatory drug is a residue of a compoundselected from the group consisting of salicylic acid, aspirin, mefenamicacid, tolfenamic acid, flufenamic acid, diclofenac, sulindac, fenbufen,indometacin, acemetacin, amfenac, etodolac, felbinac, ibuprofen,flurbiprofen, ketoprofen, naproxen, pranoprofen, fenoprofen, tiaprofenicacid, oxaprozin, loxoprofen, alminoprofen, zaltoprofen, piroxicam,tenoxicam, lornoxicam, meloxicam, tiaramide, tolmetin, diflunisal,acetaminophen, floctafenine, tinoridine and actarit.(5) The hyaluronic acid derivatives according to any one of theabove-described (1) to (4), wherein the spacer is a compound having atleast one functional group which binds to the hyaluronic acid and onefunctional group which binds to the anti-inflammatory drug.(6) The hyaluronic acid derivatives according to any one of theabove-described (1) to (5), wherein the spacer is selected from adiaminoalkane having from 2 to 18 carbon atoms, an aminoalkyl alcoholhaving from 2 to 12 carbon atoms which may have a substituent(s), and anamino acid.(7) The hyaluronic acid derivatives according to any one of theabove-described (1) to (6), wherein the hyaluronic acid has a weightaverage molecular weight of from 500,000 to 3,000,000.(8) The hyaluronic acid derivatives according to any one of theabove-described (1) to (7), wherein the anti-inflammatory drug isintroduced at a ratio of from 5 to 50 mol % per repeating disaccharideunit of hyaluronic acid.(9) A hyaluronic acid derivatives in which a non-steroidalanti-inflammatory drug is bound to hyaluronic acid through a covalentbond, which has a partial structure of hyaluronic acid disaccharide unitinto which the anti-inflammatory drug is introduced is represented bythe following formula (1):

Y—CO—NH—R¹—(O—R²)_(n)  (1)

wherein Y—CO— represents one residue of the hyaluronic acid disaccharideunit;

R² represents a non-steroidal anti-inflammatory drug residue representedby Z—CO— or hydrogen atom, with the proviso that all R²'s are nothydrogen atoms;

—HN—R¹—(O—)_(n), represents a spacer residue in a spacer compoundrepresented by H₂N—R¹—(OH)_(n) having n numbers of a hydroxyl group;

R¹ represents a linear or branched hydrocarbon group having from 2 to 12carbon atoms which may have a substituent;

—CO—NH— represents an amide bond of a carboxyl group in glucuronic acidas a constituting saccharide of the hyaluronic acid with an amino groupin the spacer compound;

—O—CO— represents an ester bond of a hydroxyl group in the spacercompound with a carboxyl group in the non-steroidal anti-inflammatorydrug residue; and

n is an integer of from 1 to 3,

wherein the hyaluronic acid derivative has a degree of substitution ofthe non-steroidal anti-inflammatory drug of from 5 to 50 mol % perrepeating disaccharide unit of hyaluronic acid, and

the carbonyl group in a hyaluronic acid residue constituting thehyaluronic acid derivative is present as an amide bond participating inthe binding with the spacer-binding anti-inflammatory drug residue or asa free carboxyl group not participating therein, according to the degreeof substitution of the non-steroidal anti-inflammatory drug residue.

(10) The hyaluronic acid derivatives according to the above-described(9), wherein the non-steroidal anti-inflammatory drug is a compoundrepresented by the following formula (2):

wherein R³ represents a substituent selected from a lower alkyl groupand a lower alkoxyl group, or a hydrogen atom;

R⁴, R⁵ and R⁶ each independently represents a substituent selected froma group consisting of lower alkyl group, a lower alkoxyl group and ahydroxyl group, a halogen atom, or a hydrogen atom; and

X's are the same or different and each represents a substituent selectedfrom a lower alkyl group and a trifluoromethyl group, or a halogen atom,and at least one of X's is a halogen atom.

(11) The hyaluronic acid derivatives according to the above-described(10), wherein the non-steroidal anti-inflammatory drug is diclofenac ora derivative thereof.(12) The hyaluronic acid derivatives according to any one of theabove-described (9) to (11), wherein R¹ in formula (1) is an ethylenegroup, a trimethylene group or a propylene group, which may have asubstituent(s).(13) The hyaluronic acid derivatives according to any one of theabove-described (1) to (12), which is obtainable by a method comprisingreacting hyaluronic acid with a spacer-bound anti-inflammatory drug, orreacting a spacer-bound hyaluronic acid with an anti-inflammatory drug,and adjusting the reaction solution to alkaline conditions.(14) The hyaluronic acid derivatives according to any one of theabove-described (1) to (13), wherein a solution obtained by dissolvingthe hyaluronic acid derivative in an aqueous medium to a concentrationof 1.0% by weight is capable of passing through a porous filter having apore size of 0.45 μm and a diameter of 25 mm, at a ratio of 2 mL perminute or more at a temperature of 24° C. under pressure of 5.0 kg/cm².(15) The hyaluronic acid derivatives according to any one of theabove-described (1) to (13), wherein a solution obtained by dissolvingthe hyaluronic acid derivative in an aqueous medium to a concentrationof 1.0% by weight is capable of passing through a porous filter having apore size of 0.22 μm and a diameter of 25 mm, at a ratio of 2 mL perminute or more at a temperature of 24° C. under pressure of 5.0 kg/cm².(16) A hyaluronic acid derivative solution which is capable of beingpushed out from an injector and which comprises the hyaluronic acidderivative according to any one of the above-described (1) to (15)dissolved in an aqueous medium.(17) The hyaluronic acid derivative solution according to theabove-described (16), wherein the aqueous medium is an aqueous mediumselected from phosphate buffered saline, saline and water for injection.(18) The hyaluronic acid derivative solution according to theabove-described (17), which is sterilized through a filter.(19) A pharmaceutical agent which comprises the hyaluronic acidderivative according to any one of the above-described (1) to (15) as anactive ingredient.(20) The pharmaceutical agent according to the above-described (19),which is an arthritis treating agent, an anti-inflammatory medicament oran analgesic.(21) The pharmaceutical agent according to the above-described (19) or(20), which is useful for parenteral administration.(22) The pharmaceutical agent according to the above-described (21),which is an injection useful for topical administration.(23) The pharmaceutical agent according to the above-described (21) or(22), which is an injection useful for intra-articular administration.(24) A pharmaceutical agent which is capable of being pushed out from aninjector and which comprises a solution in which the hyaluronic acidderivative according to any one of the above-described (1) to (15), asan active ingredient, is dissolved in an aqueous medium.(25) A kit for injection of a hyaluronic acid derivative, whichcomprises the hyaluronic acid derivative solution according to any oneof the above-described (16) to (18) which is filled in an injectorcapable of pushing out the solution.(26) The kit according to the above-described (25), wherein the filledsolution is the pharmaceutical agent according to any one of theabove-described (19) to (24).(27) A medical injection kit which is sealed with a plunger formedicament extrusion in such a manner that it can be slid and whichcomprises a syringe filled with a solution in which the hyaluronic acidderivative according to any one of the above-described (1) to (15) isdissolved in pharmaceutically acceptable phosphate buffered saline,saline or water for injection.(28) A derivative in which a spacer having a biodegradable region isbound with an anti-inflammatory drug via a covalent bond.(29) The derivative according to the above-described (28), wherein thespacer having a biodegradable region is a residue of a diaminoalkane, anaminoalkyl alcohol or an amino acid.(30) The derivative according to the above-described (28) or (29),wherein the spacer having a biodegradable region is a residue of acompound capable of binding two or more anti-inflammatory drugs to onemole of the spacer.(31) The derivative according to any one of the above-described (28) to(30), wherein the anti-inflammatory drug is a residue of a compoundselected from the group consisting of salicylic acid, aspirin, mefenamicacid, tolfenamic acid, flufenamic acid, diclofenac, sulindac, fenbufen,indometacin, acemetacin, amfenac, etodolac, felbinac, ibuprofen,flurbiprofen, ketoprofen, naproxen, pranoprofen, fenoprofen, tiaprofenicacid, oxaprozin, loxoprofen, alminoprofen, zaltoprofen, piroxicam,tenoxicam, lornoxicam, meloxicam, tiaramide, tolmetin, diflunisal,acetaminophen, floctafenine, tinoridine and actarit.(32) The derivative according to any one of the above-described (28) to(31), wherein the covalent bond is an ester bond or an amide bond.(33) The derivative according to the above-described (32), which isrepresented by the following formula (3):

H₂N—R¹—(O—R²)_(n)  (3)

wherein R² represents a hydrogen atom or a non-steroidalanti-inflammatory drug residue represented by Z—CO—, with the provisothat all R²'s are not hydrogen atoms;

H₂N—R¹—(O—)_(n) represents a spacer residue in a spacer compoundrepresented by H₂N—R¹—(OH)_(n) having n numbers of hydroxyl group;

R¹ represents a linear or branched hydrocarbon group having from 2 to 12carbon atoms which may have substituents;

—O—CO— represents an ester bond consisting of a hydroxyl group in thespacer compound and a carboxyl group in the non-steroidalanti-inflammatory drug residue; and

n is an integer of from 1 to 3.

(34) A process for producing a hyaluronic acid derivative whichcomprises hyaluronic acid bound to an anti-inflammatory drug through acovalent bond via a spacer having a biodegradable region, said processcomprising:

reacting hyaluronic acid with a spacer-bound anti-inflammatory drug, or

reacting a spacer-bound hyaluronic acid with an anti-inflammatory drug.

(35) The process for producing a hyaluronic acid derivative according tothe above-described (34), which comprises treating a solution of areaction product of hyaluronic acid with a spacer-boundanti-inflammatory drug or a solution of a reaction product of aspacer-bound hyaluronic acid with an anti-inflammatory drug underalkaline conditions.

ADVANTAGE OF THE INVENTION

According to the present invention, hyaluronic acid derivatives in whichan anti-inflammatory drug is bound to hyaluronic acid through a covalentbond via a spacer having a biodegradable region, particularly anon-steroidal anti-inflammatory drug-introduced hyaluronic acidderivative in which a non-steroidal anti-inflammatory drug is boundthrough a covalent bond (hereinafter referred to as “substance 1 of thepresent invention”), also a disease-modifying anti-rheumaticdrug-introduced hyaluronic acid derivative in which a disease-modifyinganti-rheumatic drug is bound through a covalent bond (hereinafterreferred to as “substance 2 of the present invention”, in thisconnection, the substance 1 of the present invention and the substance 2of the present invention are also called “substance of the presentinvention” as a whole), and a pharmaceutical agent which comprises oneof these derivatives as the active agent (hereinafter referred to as“pharmaceutical agent of the present invention”) are provided. Since thesubstance of the present invention is sufficiently dissolved in abuffer, saline, water for injection or the like which is used as thesolvent of injections or the like, it can be used as an injection thatcan be directly administered to the affected site. In addition, thepharmaceutical agent of the present invention can be used in thetreatment of arthritis, suppression of inflammation and suppression ofpain, and its parenteral administration or topical administration asinjections (e.g., intraarticular administration) is also possible.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing pain scores on the bradykinin-induced painmodel in rat.

FIG. 2 is a graph showing pain scores on the 1% silver nitratesolution-induced pain model in rat.

FIG. 3 is a graph showing a weight loading ratio (%) on the 1% silvernitrate solution-induced pain model in rat.

FIG. 4 is a graph showing a residual ratio in rabbit knee joint withtime by the administration of aminopropanol-ketoprofen-introduced sodiumhyaluronate (KP-HA), a mixture of ketoprofen and HA, and ketoprofen torabbit knee joint.

FIG. 5 is a graph showing effect of aminopropanol-diclofenac-introducedsodium hyaluronate having different degree of substitution (DS) on the1% silver nitrate solution induced pain model in rat.

FIG. 6 is a graph showing effect of oral administration of diclofenacsodium on the 1% silver nitrate solution induced pain model in rat.

FIG. 7 is a graph showing effects of diclofenac single drug andhyaluronic acid on the 1% silver nitrate solution induced pain model inrat.

FIG. 8 is a graph showing comparison of the effects ofaminopropanol-diclofenac-introduced sodium hyaluronate (65 kDa),diaminopropane-diclofenac-introduced sodium hyaluronate, andaminoethanol-diclofenac-introduced sodium hyaluronate on the 1% silvernitrate solution induced pain model in rat.

FIG. 9( a) is a graph showing effects (in vitro) of diclofenac sodiumsingle drug and diclofenac-introduced hyaluronic acid derivatives onCOX-2.

FIG. 9( b) is a graph showing effects (in vitro) of sodium hyaluronatesingle drug and diclofenac-introduced hyaluronic acid derivatives onCOX-2.

FIG. 10 (a) is a graph showing effect of the administration ofdiclofenac-introduced hyaluronic acid derivative on adjuvant-injectedpaw on the adjuvant-induced arthritis (AIA) model in rat.

FIG. 10 (b) is a graph showing effect of the administration ofdiclofenac-introduced hyaluronic acid derivative onadjuvant-non-injected paw on the adjuvant-induced arthritis (AIA) modelin rat.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described below based on embodiments of thepresent invention.

The substance of the present invention is a hyaluronic acid derivativein which an anti-inflammatory drug is bound to hyaluronic acid through acovalent bond via a spacer having a biodegradable region. According tothe present invention, the anti-inflammatory drug is selected fromnon-steroidal anti-inflammatory drugs (NSAID or NSAIDs) anddisease-modifying anti-rheumatic drugs (DMARD).

In this connection, the terminology “NSAIDs” generally means more thanone non-steroidal anti-inflammatory drugs, in which two or more drug areclassified, and “NSAID” means each non-steroidal anti-inflammatory drugin some cases, but they are not strictly differentiated in thisdescription.

The substance 1 of the present invention is a hyaluronic acidderivatives in which a non-steroidal anti-inflammatory drug is bound viaa covalent bond, and its structure is conceptually represented by thefollowing formula (4):

HA-SP-NSAID  (4)

wherein HA represents a hyaluronic acid chain; SP represents a spacerresidue; NSAID represents a non-steroidal anti-inflammatory drugresidue, and — represents a covalent bond.

In addition, the substance 2 of the present invention is a hyaluronicacid derivative in which a disease-modifying anti-rheumatic drug isbound via a covalent bond, and its structure is conceptually representedby the following formula (5):

HA-SP-DMARD  (5)

wherein HA represents a hyaluronic acid chain; SP represents a spacerresidue; DMARD represents a disease-modifying anti-rheumatic drugresidue; and — represents a covalent bond.

The substance of the present invention can be dissolved in an aqueoussolvent, and it is a viscous solution.

The term “aqueous solvent” as used herein means water, a buffer solutioncontaining water, and an aqueous solution or a buffer solutioncontaining a pharmaceutically acceptable metal salt, a pH adjustingagent or the like. The specific examples include water for injection,phosphate buffered saline, saline and the like.

The hyaluronic acid to be used in the substance of the present inventionis not particularly limited, so long as it is a glycosaminoglycan whichconsists of a disaccharide unit consisting of N-acetyl-D-glucosamine andD-glucuronic acid bound through a β1,3 bond as the basic core structureand is constructed by repeating β1,4 bond of the disaccharide unit,namely a generally used hyaluronic acid. In addition, it is possible touse those which are derived from animals or microorganisms or chemicalsynthesis.

The weight average molecular weight of hyaluronic acid is notparticularly limited, but from 10,000 to 5,000,000 can be exemplified.Preferably from 500,000 to 3,000,000, and more preferably from 600,000to 1,500,000 and from 1,500,000 to 3,000,000 as standards used in anarthritis treating agent and can be exemplified.

In this connection, the hyaluronic acid to be used in the presentinvention may be either in a free form of not forming a salt or apharmaceutically acceptable salt. The pharmaceutically acceptable saltof hyaluronic acid includes salts with alkali metal ions such as asodium salt, a potassium salt, and salts with alkaline earth metal ionssuch as a magnesium salt, and a calcium salt. When a hyaluronic acidderivative is used in a pharmaceutical preparations or the like for usein the living body, the hyaluronic acid salt to be used is preferably asalt with an alkali metal ion, particularly a salt with a sodium ion,because of its high affinity for the living body.

The NSAIDs as one of the anti-inflammatory drugs concerned in thepresent invention generally mean the whole compounds which are usuallycalled non-steroidal anti-inflammatory agents and are not particularlylimited, but those which are applied to arthritis are particularlypreferable. As a conventional classification method of NSAIDs, there isa classification based on the difference of its core structure inchemical structure. When the NSAIDs to be applied to the presentinvention are exemplified based on this classification, salicylic acidtype NSAIDs include salicylic acid, aspirin and the like; fenamic acidtype NSAIDs include mefenamic acid, tolfenamic acid, flufenamic acid andthe like; aryl acetate type NSAIDs include diclofenac, sulindac,fenbufen, indometacin, acemetacin, amfenac, etodolac, felbinac and thelike; propionic acid type NSAIDs include ibuprofen, flurbiprofen,ketoprofen, naproxen, pranoprofen, fenoprofen, tiaprofenic acid,oxaprozin, loxoprofen, alminoprofen, zaltoprofen and the like; oxicamtype NSAIDs include piroxicam, tenoxicam, lornoxicam, meloxicam and thelike; and other NSAIDs include tiaramide, tolmetin, diflunisal,acetaminophen, floctafenine, tinoridine and the like.

As the NSAIDs to be applied to the present invention, those which have afunctional group such as a carboxyl group, a hydroxyl group or an aminogroup in the chemical structure are preferable. Since it is possible toselect functional group of the spacer according to the functional groupsof these NSAIDs, the substance 1 of the present invention is notparticularly limited, but the NSAIDs which at least have a carboxylgroup are most preferably used.

Among these, compounds which have the core structure represented by thefollowing formula (6) are more preferably used:

Furthermore, the compounds represented by the following formula (2) areparticularly preferably used:

R³ represents a substituent selected from lower alkyl groups and loweralkoxyl groups, or a hydrogen atom; R⁴, R⁵ and R⁶ each independentlyrepresents a substituent selected from a lower alkyl group, a loweralkoxyl group and a hydroxyl group, a halogen atom, or a hydrogen atom;and X's are the same or different each other, and each independentlyrepresents a substituent selected from a lower alkyl group and atrifluoromethyl group, or a halogen atom, wherein at least one of X's isa halogen atom. In addition, the above-described lower alkyl group andlower alkoxyl group are preferably a lower alkyl group and a loweralkoxyl group having from 1 to 12 carbon atoms which are allowed to bebranched, and more preferably a lower alkyl group and a lower alkoxylgroup having from 1 to 6 carbon atoms which are allowed to be branched.

Also, when a carboxymethyl group and an amino residue are positioned atthe 1-position and 2-position, respectively, of the benzene ring towhich R³ is bound, R³ is preferably bound to the 5-position.

As the compounds represented by the above-described formula (2), forexample, the compounds described in WO 99/11605 can be cited, and thecontents described therein are incorporated herein by reference.Carboxyl group in the NSAIDs is not limited to free form but also tosalt form.

The DMARD as the other one of the anti-inflammatory drugs concerned inthe present invention generally mean whole pharmaceutical preparationsusually used as anti-rheumatic agents and are not particularly limited,but those which have a functional group, such as a carboxyl group, ahydroxyl group, an amino group or a mercapto group in the chemicalstructure are preferable. The DMARD includes actarit, methotrexate,salazosulfapyridine, bucillamine and the like.

In this connection, the above-described NSAID and DMARD can be cited asthe anti-inflammatory drugs concerned in the present invention, but thecompounds which have carboxyl group are particularly preferable.

In this connection, it is possible to introduce those functional groupsto hyaluronic acid via a desired binding mode by selecting functionalgroup of the spacer moiety depending on the functional groups owned bythe above-described NSAIDs and DMARD. In addition, it is not alwaysnecessary that one species of NSAID or DMARD is introduced into thesubstance of the present invention, and hyaluronic acid derivatives towhich two or more species of NSAID and DMARD are introduced are alsoincluded therein.

The above-described spacer represented by SP is a spacer which has aregion that can be biodegraded and is the residue of a compound havingat least one functional group which binds to hyaluronic acid and onefunctional group which binds to NSAIDs or DMARD (hereinafter alsoreferred to as “spacer compound”). The region of the spacer which can bebiodegraded is not particularly limited, so long as the NSAIDs or DMARDreleased from the hyaluronic acid derivatives have the effect, but it ispreferable that the region is cleaved at the binding region of NSAIDs orDMARD with the spacer.

Respective functional groups of the spacer compound can be optionallyselected depending on the binding modes with hyaluronic acid and NSAIDsor DMARD. For example, when spacer molecule is introduced at thecarboxyl group of the hyaluronic acid through amide bond, a spacercompound with amino group can be selected, and in the case of ester bondat the carboxyl group of the hyaluronic acid, spacer with hydroxyl groupcan be selected. If the spacer is introduced at the hydroxyl group ofthe hyaluronic acid through ester bond, spacer with carboxyl group canbe selected. In this case, from the viewpoint of the conciseness for theintroduction of spacer molecule into hyaluronic acid and the stabilityin the living body, a spacer compound having amino group which can beintroduced into the carboxyl group of hyaluronic acid through amido bondcan be cited as one of the preferable embodiments.

In the same manner, the functional group of a spacer compound whichbinds to NSAIDs or DMARD can also be selected based on the functionalgroup owned by NSAIDs or DMARD. For example, in the case of NSAIDs orDMARD having hydroxyl group, it can be bound through an ester bond whena spacer compound having carboxyl group is selected, in the case ofNSAIDs or DMARD having carboxyl group, it can be bound through an esterbond when a spacer compound having hydroxyl group is selected, or can bebound through an amide bond when a spacer compound having amino group isselected, and in the case of NSAIDs or DMARD having mercapto group, itcan be bound through thioester bond when a spacer compound havingcarboxyl group is selected.

In this case, when the aptness to be biodegraded is taken intoconsideration, a spacer compound having a functional group which canbind through an ester bond to the carboxyl group of NSAIDs or DMARD ispreferable, and it is particularly preferable that the carboxyl group ofNSAIDs or DMARD and the hydroxyl group of the spacer compound are boundthrough an ester bond.

As described above, it is possible to select spacer compound optionallyin accordance with the characteristics of hyaluronic acid and NSAIDs orDMARD, but, for example, a diaminoalkane having from 2 to 18 carbonatoms, an aminoalkyl alcohol having from 2 to 12 carbon atoms which mayhave a substituent(s), an amino acid and the like can be exemplified.The amino acid may be a naturally occurring or non-naturally occurringamino acid and is not particularly limited, but preferably, glycine,β-alanine and γ-aminobutyric acid can be exemplified.

As described above, when the binding mode of hyaluronic acid with NSAIDsis taken into consideration, an aminoalkyl alcohol having from 2 to 12carbon atoms which may have a substituent can be cited as a preferableexample of the spacer compound.

In addition, it may be a spacer compound which has two or more of thesefunctional groups capable of binding to NSAIDs or DMARD, in one molecule(hereinafter also referred to as “multivalent spacer compound”).

When a multivalent spacer compound is selected, two or more of NSAIDs orDMARD can be bound simultaneously to one spacer. Accordingly, two ormore of NSAIDs or DMARD can be introduced simultaneously into thefunctional group, for example, one carboxyl group, of hyaluronic acid towhich the NSAIDs or DMARD are to be introduced. Examples of thesemultivalent spacer compounds include serinol and a derivative thereof, aserine derivative, a threonine derivative, 2-amino-1,5-pentanediol and aderivative thereof, 3-amino-1,2-propanediol and a derivative thereof,tris(hydroxymethyl)aminomethane and a derivatives thereof, bishomotrisand a derivatives and the like.

The merit of using this multivalent spacer compound is that more largeramount of NSAIDs or DMARD can be introduced without allowing a largenumber of carboxyl groups and hydroxyl groups contributing to thehydrophilic property of hyaluronic acid to the substitution reaction, sothat hydrophilic property, namely solubility in the aqueous medium canbe kept in despite the large amount of NSAIDs or DMARD are introduced inthe molecule.

The method for synthesizing the substance of the present invention isnot particularly limited, so long as it is a method by which the solublesubstance of the present invention as described above can be obtained.

In this connection, in the case of a hyaluronic acid derivative in whicha compound is introduced into hyaluronic acid, the carboxyl group andhydroxyl group owned by hyaluronic acid generally take part in thebinding to the compound, so that hydrophilic property of the hyaluronicacid derivative decreases as the degree of substitution of the substanceincreases.

An example of the method for synthesizing the substance 1 of the presentinvention include a method which comprises carrying out an alkalitreatment after an introduction of NSAIDs into hyaluronic acid via aspacer having a region capable of being biodegraded.

The above-described method of alkali treatment after the introductionreaction in order to make the reaction solution alkaline is notparticularly limited, so long as it is a treatment by which the solutionbecomes alkaline. Specifically, a method in which either an organic baseor an inorganic base is added to the solution can be exemplified, but aninorganic base is preferable when the treatment thereafter and the likeare taken into consideration. In addition, even among inorganic bases,weaker base such as sodium hydrogen carbonate or sodium carbonate ispreferable rather than a stronger base such as sodium hydroxide, due tothe lower influence on hyaluronic acid and NSAIDs. As the pH conditionsof alkali treatment in this case, from 7.2 to 11, preferably from 7.5 to10, can be exemplified.

The treating time of the alkali treatment is not particularly limited,so long as it does not exert influence on molecular weight reduction ofhyaluronic acid, but from 2 to 12 hours, preferably from 2 to 6 hours,can be cited, and a soluble hyaluronic acid derivative can be obtainedwithout exerting influence on hyaluronic acid when the treatment iscarried out for the time period.

As a specific example, the intended soluble hyaluronic acid derivativecan be obtained by allowing a spacer-introduced NSAIDs derivative toreact with hyaluronic acid, adding a weak alkali such as sodium hydrogencarbonate to the reaction solution, followed by stirring for severalhours, and then carrying out post-treatments such as neutralization,ethanol precipitation and drying.

The method described above can also be applied to the synthesis of thesubstance 2 of the present invention, so that a soluble substance 2 ofthe present invention can be obtained.

In this connection, the method for introducing a spacer and NSAIDs orDMARD into hyaluronic acid may be either a method in which the spacer isintroduced into hyaluronic acid, and then NSAIDs or DMARD is introducedinto the spacer-linked hyaluronic acid or a method in which a spacer isintroduced into NSAIDs or DMARD in advance, and then the spacer-linkedNSAIDs or the spacer-linked DMARD is introduced into hyaluronic acid,but the latter method is preferable.

The method for respectively binding NSAIDs or DMARD, hyaluronic acid andspacer is not particularly limited, but it is possible to use agenerally used conventional method as a means for carrying out thebinding reaction with the proviso that it is a method that can attainester bond formation, amide bond formation, thioester bond formation andthe like. And The reaction conditions can be optionally judged andselected by one skilled in the art.

In this connection, the condensation of hyaluronic acid with thespacer-linked NSAIDs or spacer-linked DMARD or with the spacer compoundcan be attained by using either the carboxyl group or hydroxyl group ofhyaluronic acid. But the carboxyl group can more easily attained thecondensation due to the higher reactivity owned by the functional group.The method for attaining such a condensation, for example, includes amethod in which a water-soluble condensing agent such as a water-solublecarbodiimide (e.g., 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDCI HCl), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidemethiodide, etc.) is used, a method in which a condensation additiveagent such as N-hydroxysuccinimide (HOSu) or N-hydroxybenzotriazole(HOBt) and the above-described condensing agent are used, an activeester method, an acid anhydride method and the like. Among these, themethod in which a water-soluble condensing agent is used, or the methodin which a condensation additive agent and a water-soluble condensingagent is used, as the reaction in the presence of an aqueous solvent, ispreferable, and the method in which a condensation additive agent and awater-soluble condensing agent is used is particularly preferable fromthe viewpoint of inhibiting side reaction. It is preferable that thecarboxyl group of hyaluronic acid is bound to the spacer-linked NSAIDsor spacer-linked DMARD or the spacer compound through an ester bond oran amide bond, more preferably through an amide bond.

It is possible to adjust the degree of substitution of NSAIDs or DMARDto hyaluronic acid regarding the substance of the present invention bychanging the amount of the condensing agent, condensation additiveagent, spacer-linked NSAIDs or spacer-linked DMARD during the processfor synthesizing the substance of the present invention. In thisconnection, the degree of substitution can be measured by measuringabsorbance or by a method which uses HPLC, NMR or the like.

According to the present invention, the degree of substitution of NSAIDsor DMARD is not particularly limited, so long as solubility of thederivative in the aqueous solvent is maintained, but from 0.1 to 80% bymol is preferable and from 5 to 50% by mol is more preferable, based onthe repeating disaccharide unit of hyaluronic acid. In addition, whenthe substance of the present invention is used as an active ingredientof a pharmaceutical preparation, the optimum degree of substitution isdetermined by taking effective concentration or sustained releaseefficiency of NSAIDs or DMARD in the affected site into consideration.

As described above, a spacer-linked NSAIDs or spacer-linked DMARD isintroduced into the carboxyl group of hyaluronic acid, the carboxylgroup forms an amide bond or an ester bond to reduce or lose itshydrophilic property.

As one of the means for solving this problem, introduction of a numberof NSAIDs or DMARD becomes possible while keeping the hydrophilicproperty, by using a multivalent spacer compound. For example, when anaminotriol derivative having 3 hydroxyl groups and 1 amino group is usedas a spacer compound, introduction of NSAIDs into all of the 3 hydroxylgroup results in the introduction of 3 molecules of NSAIDs into 1 spacermolecule. When this aminotriol-linked NSAIDs is introduced into thecarboxyl group of hyaluronic acid, for example, at a degree ofsubstitution (degree of substitution based on hyaluronic disaccharideunit) of 20%, it means that the degree of substitution of NSAIDs is 60%equivalent to 3 fold higher of the aminotriol-linked NSAIDs's degree ofsubstitution.

In addition, as described above, solubility of the hyaluronic acidderivative in the aqueous medium is maintained when the method in whichan alkali treatment is carried out after the introduction reaction forsynthesizing an anti-inflammatory drug-introduced hyaluronic acidderivative, which was cited as an example of the method for synthesizingthe substance of the present invention, is employed. This solubilitykeeping effect is markedly useful, because it is not so necessary toconsider kind of the spacer compound, nor the degree of substitution ofa medicament and the like, and the treatment is convenient.

In summarizing the above-described explanations, as a specificallypreferable embodiment of the substance 1 of the present invention, ahyaluronic acid derivative having a disaccharide unit constitutinghyaluronic acid represented by the following formula (1) can be forexample cited. In this connection, the following formula (1) shows apartial structure per disaccharide unit constituting hyaluronic acidwherein an anti-inflammatory drug-introduced N-acetyl-D-glucosamine andD-glucuronic acid are bound via a β-1,3 bond.

Y—CO—NH—R¹—(O—R²)_(n)  (1)

wherein Y—CO— represents one residue of the disaccharide unitconstituting hyaluronic acid; R² represents an NSAID residue representedby Z—CO— or a hydrogen atom, in which all R²s are not hydrogen atoms;—HN—R¹—(O)_(n) represents a spacer residue in a spacer compoundrepresented by H₂N—R¹—(OH)_(n) having n numbers of hydroxyl group; R¹represents a linear or branched hydrocarbon group having from 2 to 12carbon atoms which may have a substituent(s); —CO—NH— represents anamide bond of a carboxyl group in glucuronic acid as a constitutingsaccharide of hyaluronic acid with an amino group in the spacercompound; —O—CO— represents an ester bond of a hydroxyl group in thespacer compound with the carboxyl group owned by NSAID; and n is aninteger of from 1 to 3. In this connection, the carbonyl group in ahyaluronic acid residue constituting the hyaluronic acid derivative ispresent as an amide bond involved in the binding with the spacer-bindinganti-inflammatory drug residue or as a free carboxyl group not involvedthereto, according to the degree of substitution of the NSAID residue.

The substituent in R¹ includes an alkyl group, an alkenyl group, an arylgroup, an alkoxy group, an acyl group, a carboxyl group, a halogen andthe like, wherein the number of carbon atoms in the alkyl group, alkenylgroup, alkoxy group and acyl group is preferably from 1 to 11, morepreferably from 1 to 4, and the phenyl group is preferable as the arylgroup. For example, serine can be exemplified as the spacer compoundhaving a carboxyl group as the substituent, and threonine as the spacercompound having a carboxyl group and a methyl group.

In this connection, according to the above-described formula (1), Y—COOHrepresents one disaccharide unit constituting hyaluronic acid before thereaction; H₂N—R¹—(OH)_(n) represents a spacer compound before thereaction; and HOOC—Z represents NSAID before the reaction.

As a most suitable method for synthesizing the hyaluronicacid-constituting disaccharide unit represented by the above-describedformula (1), a method in which a spacer compound and NSAID are bound andthen allowed to react with hyaluronic acid can be exemplified.Conceptual expression of this reaction is as follows.

R⁷HN—R¹(OH)_(n)+HOOC—Z→(esterformation)→(deprotection)→H₂N—R¹—(O—R²)_(n)H₂N—R¹—(O—R²)_(n)+Y—COOH→Y—CO—NH—R¹—(O—R²)_(n)

R⁷ represents a protecting group of an amino group, wherein theprotecting group is not particularly limited, because protecting groupsgenerally used as the protecting group of an amino group can be used,and examples include a urethane type protecting group such as atert-butoxycarbonyl group, a benzyloxycarbonyl group and a9-fluorenylmethyloxycarbonyl group, and an acyl type protecting groupsuch as a formyl group and a phthaloyl group, and a urethane typeprotecting group is preferable. In this connection, R¹, R² and Z are asdefined above.

However, the above description is a conceptual explanation of thereaction pathway, and a design and the like for efficiently carrying outthe reaction, which can be deduced by those skilled in the art, areomitted herein.

In the above-described formula (1), R¹ is more preferably a linear orbranched chain hydrocarbon group having from 2 to 5 carbon atoms whichmay have a substituent(s), particularly preferably a hydrocarbon grouphaving 2 or 3 carbon atoms, and examples include an ethylene group, atrimethylene group and a propylene group.

Also, as the NSAID to be used in the above-described formula (1), it ispossible to select it from the above-described NSAID. In addition,compounds represented by the following formula (7) can be preferablyexemplified.

R⁸ represents a substituent selected from a lower alkyl groups and alower alkoxyl groups, or a hydrogen atom, and is more preferably a loweralkyl group having from 1 to 12 carbon atoms which may have a branch, ora hydrogen atom, and particularly preferably a lower alkyl group havingfrom 1 to 4 carbon atoms or a hydrogen atom.

X¹ and X² each independently represents a substituent selected fromlower alkyl groups and a trifluoromethyl group or a halogen atom,wherein at least one of them is a halogen atom. X¹ and X² are preferablyhalogen atoms which are the same or different, and more preferablyselected from a fluorine atom and a chlorine atom.

In addition, it is preferable that R⁸ is bound to the 5-position of thebenzene ring to which R⁸ is bound, when the carboxymethyl group and theamino group are positioned at the 1-position and the 2-position in thebenzene ring, respectively.

Specific examples of the compounds represented by the above-describedformula (7) include compounds represented by the following formulae (8)and (9):

For example, when a diclofenac-introduced hyaluronic acid derivative issynthesized using the diclofenac represented by the formula (9), the—CO—Z in the above-described formula (1) is represented by the followingformula (10):

In this connection, the diclofenac-introduced hyaluronic acidderivatives have very strong analgesic action and anti-inflammatoryaction.

As the hyaluronic acid which can be used in the substance of the presentinvention having the disaccharide unit constituting hyaluronic acidrepresented by the above-described formula (1), preferably a hyaluronicacid having a weight average molecular weight of from 50,000 to3,000,000, more preferably a hyaluronic acid having a weight averagemolecular weight of from 50,000 to 2,000,000, is selected.

The degree of substitution of NSAIDs (DS) in the substance of thepresent invention having the disaccharide unit constituting hyaluronicacid represented by the above-described formula (1) is preferably from 5to 50% by mol, more preferably from 10 to 50% by mol, based on therepeating disaccharide unit of hyaluronic acid.

As a significant characteristic of the substance of the presentinvention, a point that the substance of the present invention can bedissolved in an aqueous solvent, namely easily water-soluble, can becited, so that when an aqueous solvent is added to the substance of thepresent invention, it dissolves without carrying out heating,solubilization treatment and the like. In this connection, even when thedegree of substitution is high, namely 5% or more, or further 10% ormore, it can be dissolved. Thus, a solution prepared by dissolving thesubstance of the present invention in an aqueous medium is an injectableliquid and has an ability to pass through a filtration filter. By theway, as described above, it is known that when a medicament havinghigher hydrophobic property, such as NSAIDs or DMARD, is introduced intohyaluronic acid having higher hydrophilic property, the product becomeswater-semi-insoluble gel having high viscoelasticity or insoluble matterbecause of the increase of hydrophobic property of the hyaluronic acidmolecule itself, so that such a product is not suitable for injectionswhich are extruded by an injector.

However, since solubility of the hyaluronic acid derivative ismaintained for example by carrying out an alkali treatment during itsproduction process as described above, the substance of the presentinvention can be a transparent solution having the ability to passthrough a filtration filter.

Thus, the solution of the substance of the present invention can besubjected to a filtration so that dust removal, removal ofmicroorganisms and sterilization of microorganisms by filtration can bepossible. That is, removal of dust and microorganisms can be effectiveby passing through a 5 μm or 0.45 μm filter, and more preferably,sterilization also becomes possible by passing through a 0.22 μm filter.

More specifically, it is preferable that a solution prepared bydissolving the substance of the present invention in an aqueous mediumto be a concentration of 1.0% by weight is capable of passing through aporous filter (pore size 0.45 μm, diameter 25 mm) at a ratio of 2 mL perminute or more at a temperature of 24° C. under pressure of 5.0 kg/cm².

Also, it is more preferable that a solution prepared by dissolving thesubstance of the present invention in an aqueous medium to be aconcentration of 1.0% by weight is capable of passing through a porousfilter (pore size 0.22 μm, diameter 25 mm) at a ratio of 2 mL per minuteor more under the same conditions described above.

As described below, when the substance of the present invention is usedas a medicament to be applied to a living body (a mammal, particularlypreferably human), dust removal, and removal of microorganisms, andsterilization of microorganisms become essential items so that such acharacteristic of the substance of the present invention is markedlyuseful. In addition, in the case of the sterilization by heating,ultraviolet ray irradiation or the like, there is a possibility ofcausing degradation, reduction of molecular weight and the like of thehyaluronic acid derivative, but such a problem can be avoided in thecase of filtration sterilization.

The pharmaceutical agent of the present invention is a pharmaceuticalpreparation comprising a hyaluronic acid derivative as the substance ofthe present invention as an active ingredient. By taking advantage ofthe above-mentioned characteristics of the substance of the presentinvention, the pharmaceutical agent of the present invention can take anembodiment in which the substance can be extruded from an injector andthe like, and is also used as a solution of the substance of the presentinvention dissolved in an aqueous medium. For example, a solution inwhich saline, phosphate buffered saline or water for injection capableof being administered to the living body is used as the solvent andwhich contains the substance of the present invention at a concentrationof from 0.1% by weight to 10% by weight can be cited. It is preferablethat this solution is not turbid but transparent.

As described above, the pharmaceutical agent of the present invention isapplicable to the dust removal, removal of microorganisms, andsterilization of microorganisms by filter filtration. Removal of dustand microorganisms becomes possible by passing through a 5 μm or 0.45 μmfilter, and sterilization also becomes possible by passing through a0.22 μm filter. In addition, it is possible also to use thepharmaceutical agent of the present invention together with thesubstance of the present invention and a pharmaceutically acceptablecarrier, within such a range that the advantage owned by thepharmaceutical agent of the present invention, namely the property to besterilized by filtration, is not spoiled.

It is preferable that the pharmaceutical agent of the present inventionprepared in this manner can be subjected to filtration sterilization andalso in such a state that it has a certain degree of viscoelasticity.

It is possible to use the pharmaceutical agent of the present inventionas a medicament for parenteral administration use or a medicament fortopical administration use. As the embodiment of using it in theparenteral administration and topical administration, a solutionprepared by dissolving the above-described substance of the presentinvention in an aqueous solvent is preferable, and administrationmethods such as injection and infusion can be preferably exemplified(according to this description, the “infusion” sometimes includes“injection”). By carrying out topical administration by infusion, sideeffects in the digestive organ system can be avoided. In addition, sincethe metabolism by the digestive organ system can also be avoided, it ispossible to reduce the dose in comparison with the case of oraladministration, and what is more, the problem of systemic toxicitycaused by a large dose of oral administration can also be avoided.

Regarding the extrusion device to be used in injection, infusion and thelike, it is possible to use implements generally used for the purpose ofadministering a filled medicament by extrusion, such as an injector andan infusion device.

In this connection, it is possible also to provide a kit in which asolution of the pharmaceutical agent of the present invention or thesubstance of the present invention is filled in an extrudable infusiondevice equipped with a plunger for medicament extrusion or the like. Inaddition, it is possible to make the kit into a medical injection kit inwhich a solution, prepared by dissolving the substance of the presentinvention in a pharmaceutically acceptable phosphate buffered saline,saline or water for injection, is filled in a syringe and sealed with aslidable plunger for medicament extrusion in such a manner. In thisconnection, it is possible to use a generally used device as the plungerfor medicament extrusion, which is formed from an elastic body such as arubber or a synthetic rubber and inserted into a syringe under a closelycontacted state in such a manner that it can be slid. In addition, aplunger rod for extruding a medicament by carrying out pushing operationof the plunger may also be included in the kit.

Although the disease to be treated and route of administration of thepharmaceutical agent of the present invention are not particularlylimited, it is possible to use it as a therapeutic agent for the purposeof treating arthritis, suppression of inflammation, suppression of painand the like (hereinafter also referred to as “therapeutic agent of thepresent invention”), which is preferable. In this connection, accordingto this description, the “therapeutic agent” includes not only a“treating agent” but also a medicament which is used for the purpose ofpreventing disease or alleviation of symptoms.

Not only the therapeutic agent of the present invention has thesustained release action of anti-inflammatory drugs such as NSAIDs andthe medicament delivery system action as described below, but also theeffect of hyaluronic acid pharmaceutical preparations currently used inthe clinical field on arthritis can also be expected at the same time,in addition to the therapeutic effect by anti-inflammatory drugs intreating arthritis.

In addition, the dose of the therapeutic agent of the present inventionis not particularly limited, because it is an item which should beindividually decided according to the route of administration,administration form, using purpose, and specific symptoms, age, bodyweight and the lie of the animal to be treated, in such a manner thatits therapeutic effect is exerted most appropriately. For example, inthe case of injections for human use, approximately from 1 mg to 1,000mg, preferably approximately from 5 mg to 500 mg, more preferablyapproximately from 10 mg to 100 mg, per adult per once, based on ahyaluronic acid derivative can be cited. However, it is considered thatstrength of the medicament effect owned by the NSAIDs or DMARD used inthe substance of the present invention as an active ingredient has greatan influence on the therapeutic agent of the present invention, so thatthe range described above is not always suitable, and it is necessary toset it by taking the dose converted into the NSAIDs or DMARD singlepreparation into consideration. In addition, as is shown in Examplesdescribed below, different from the case in which NSAIDs singlepreparation is administered, the pharmaceutical agent of the presentinvention is present in the administered site stably and continuously,so that it is necessary to set it by also taking this point intoconsideration.

The site to which the therapeutic agent of the present invention is tobe applied is not particularly limited, so long as it is an applicationsite by parenteral administration, and joints are preferable among theparts, and a knee joint, a shoulder joint, a hip joint, a jaw joint andthe like are particularly preferable. Especially, application toosteoarthritis of knee (OA) and rheumatoid arthritis of knee (RA) ispreferable.

In this connection, when the pharmaceutical agent of the presentinvention is used as a therapeutic agent for arthritis, a properconcentration as joint infusions (injections) can be optionally selectedas described above, and the concentration of solution is preferably from0.3 to 3.0% by weight, more preferably from 0.5 to 1.5% by weight.

As one of the most preferable embodiments of the pharmaceutical agent ofthe present invention, the following construction can be cited.

NSAID:

-   -   Compound represented by the above-described formula (2)

Spacer and Binding Mode:

-   -   Aminoalkyl alcohol is bound with NSAID via an ester bond, bound        with hyaluronic acid via an amide bond

Molecular Weight of Hyaluronic Acid:

-   -   Weight average molecular weight: 500,000 to 3,000,000

Degree of Substitution of NSAID:

-   -   5 to 50% by mol per hyaluronic acid disaccharide unit

Concentration and Solvent:

-   -   Phosphate buffered saline having a concentration of from 0.3 to        3.0% by weight

Providing Condition:

-   -   Filled in a syringe under sterilized state.

In addition, as the NSAID, the compound represented by theabove-described formula (7) is more preferable, and the compoundsrepresented by the above-described formula (8) and the above-describedformula (9) are further preferable, and diclofenac or a derivativethereof is particularly preferable. As the spacer, it is more preferablywhen selected from aminopropyl alcohol or aminoethyl alcohol.

As the degree of substitution, from 10 to 50% by mol per hyaluronic aciddisaccharide unit is more preferable.

Also, it is most preferable when filtration through a 5 μm or 0.45 μmfilter is possible, and further when filtration through a 0.22 μm filteris possible.

As shown in the examples which are described below, it is particularlysuitable to use the pharmaceutical agent of the present invention as atherapeutic agent for arthritis, particularly as joint infusions forarthritis treatment. For example, when low molecular weight compoundssuch as NSAIDs are directly infused into joint cavity, these compoundsare immediately removed into blood stream through synovium, so that agreater effect cannot be expected.

On the other hand, when a solution of an NSAID s-introduced hyaluronicacid derivative to which NSAIDs as the substance of the presentinvention are introduced through a covalent bond is administered intojoint cavity, NSAIDs are continuously present in the synovium tissue asis shown later in the examples, while the low molecular weight compoundalone is quickly metabolized in the synovium. As is generally known,hyaluronic acid has affinity for synovium. For the reason it isconsidered that the pharmaceutical agent of the present invention isretained to a certain degree in the synovium under a state in whichhyaluronic acid and NSAIDs are bound, and after gradually incorporatedinto tissues or cells, NSAIDs are released from hyaluronic acid and takethe action. That is, in the case of the administration of thepharmaceutical agent of the present invention, NSAIDs are notimmediately removed into blood stream, but NSAIDs are persistentlypresent in the joint fluid and synovium, so that it shows persistenteffect.

Based on this, it is preferable that the binding of hyaluronic acid witha spacer compound in the pharmaceutical agent of the present inventionshows resistance to its biodegradation in comparison with the binding ofNSAIDs with the spacer compound. In addition, preferred is an embodimentin which the binding site of NSAIDs with the spacer compound is notdegraded in the joint cavity, but degraded in the synovium tissue afterincorporated into the synovium. By changing binding modes of NSAIDs withthe spacer compound and hyaluronic acid with the spacer compound,resistance to the biodegradation can be changed thereby renderingpossible control of the aptness to release and the releasing ratio. Forexample, when hydrolysis occurring in the living body is considered, anester bond is more susceptible against degradation than amide bond.Thus, in selecting a spacer which binds to hyaluronic acid via an amindebond and NSAIDs via an ester bond, the ester bonds are susceptible tohydrolysis and NSAIDs are released from the substance of the presentinvention which has been hydrolyzed to be active. A pharmaceuticalpreparation for sustained release use is also possible by thepharmaceutical agent of the present invention.

In this connection, in the examples which are described below, when 2kinds of the substance of the present invention in which hyaluronic acidwas bound to a spacer compound via an amide bond and NSAIDs was bound tothe spacer compound via amide bond or ester bond were respectivelyadministered, the substance of the present invention in which NSAIDs andthe spacer compound were bound via an ester bond showed more significantpain suppressing effect.

It is known that inhibition of prostaglandin production by thecyclooxigenase (COX) inhibitory activity in the target cell plays a roleas the mechanism of NSAIDs which suppress inflammation and painaccompanied by arthritis. Evaluation of the substance of the presentinvention was carried out by using Chemiluminescent COX InhibitorScreening Assay Kit (manufactured by Cayman) which is a method generallyused for the evaluation of COX-2 inhibitory activity. As a result, theCOX-2 inhibitory activity was not found in the substance 1 of thepresent invention, by the dose by which NSAIDs as single preparationclearly showed the COX-2 inhibitory activity and also by the dose of thesubstance 1 of the present invention containing an amount of NSAIDswhich corresponds to the dose of the single preparation converted fromNSAIDs.

This in vitro results can not be extended to the living body which isconcerted by various conditions and states, however, it is easilyspeculated that the release of NSAIDs in the acting area by thepharmaceutical agent of the present invention is preferable.

In addition, the substance of the present invention is also useful as abase material of the drug delivery system (DDS) of NSAIDs or DMARDwhich, being low molecular weight compounds, are known to be difficultin effective delivery to the target site (cells) by single drugadministration because of the quick metabolism in the living body. Inorder to obtain efficient results by reducing influence of themetabolism, it is markedly important to deliver NSAIDs or DMARD to thetarget cells in the form of the NSAIDs- or DMARD-introduced hyaluronicacid derivative of the present invention and to further incorporate intothe cells in the same form to effect persistent presence in the targetsite.

The amount of a medicament effective for the treatment at theadministered region can be efficiently kept by the use of the substanceof the present invention, in comparison with single administration ofthe medicament, so that much stronger therapeutic effect can be expectedwith much smaller dose by oral administration. In addition, sincesustained release ability and persistency of the effect can be improved,reduction of the number of times of administration and the like in theclinical use can also be expected.

EXAMPLES

The present invention is described below more specifically based onExamples. However, there is no intention to limit the technical scope ofthe present invention by this.

In this connection, all of the hyaluronic acid and sodium hyaluronateused in the following Examples were purchased from SeikagakuCorporation.

Hereinafter, as the phosphate buffered saline (PBS), 5 mM PBS was usedin the following Examples, unless otherwise indicated.

Test Example Filter Pass Through Test

PBS in which each substance to be tested was dissolved to aconcentration of 1.0% by weight was prepared. At a temperature of 24° C.under pressure of 5.0 kg/cm², each solution of the substances to betested prepared in the following examples was passed through a 0.45 μmporous filter (25 mm in diameter), and the passed amount (ml) per 1minute was measured A case in which 2 ml or more was passed is shown by“A”, and a case in which less than 2 ml was passed by “B”, and a case inwhich none passed by “C”.

Production Examples Reference Example 1 Synthesis oft-butoxycarbonyl-aminopropanol (Boc-NH(CH₂)₃OH) (Boc-aminopropanol)

In 10 ml of dichloromethane, 1.542 g (20.5 mmol) of aminopropanol wasdissolved, and 10 ml of a 4.484 g (20.5 mmol) di-t-butyl dicarbonate(Boc₂O)/dichloromethane solution was slowly added dropwise thereto underice-cooling. Thereafter, the reaction solution was returned to roomtemperature and stirred for 2 hours and 40 minutes, disappearance of thestarting materials was confirmed by thin layer chromatography (TLC), andthen dichloromethane was evaporated under reduced pressure. The reactionquantitatively progressed, and an oily substance was obtained at a yieldof 3.92 g. The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.46 (9H, s, Boc), 1.66 (2H, quant,—NHCH₂ CH₂ CH₂O—), 3.27 (3H, m, —NHCH₂ CH₂CH₂O—), 3.66 (2H, m, —NHCH₂CH₂CH₂ O—), 4.91 (1H, br, CH₂ OH)

Example 1 Synthesis of Aminopropanol-Ketoprofen Hydrochloride 1)Synthesis of Boc-Aminopropanol-Ketoprofen

In 14 ml of dichloromethane, 2.371 g (13.5 mmol) of Boc-aminopropanoland 3.441 g (13.5 mmol) of ketoprofen (manufactured by Tokyo KaseiKogyo) were dissolved, and 323 mg (2.6 mmol) of 4-dimethylaminopyridine(DMAP) and 2.833 g (14.8 mmol) of water-soluble carbodiimidehydrochloride (WSCI.HCl)/14 ml dichloromethane were added thereto inthis order under ice-cooling. After returning to room temperature andstirring overnight, dichloromethane was evaporated under reducedpressure, and ethyl acetate was added thereto, followed by separation bywashing with 5% citric acid twice, water, 5% sodium hydrogen carbonatetwice, water and saturatedbrine consecutively. After dehydration dryingwith sodium sulfate, ethyl acetate was evaporated under reduced pressureto give 5.430 g of the titled compound (yield 98%). The structure wasidentified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.43 (9H, s, Boc), 1.54 (3H, d,—OCOCH(CH₃ )—), 1.77 (2H, quant, —NHCH₂ CH₂ CH₂O—), 3.09 (2H, m, —NHCH₂CH₂CH₂O—), 3.82 (1H, q, —OCOCH(CH₃)—), 4.15 (2H, m, —NHCH₂CH₂ CH₂ O—),4.69 (1H, br, —NHCH₂—), 7.42-7.83 (9H, m, Aromatic H)

2) Synthesis of Aminopropanol-Ketoprofen Hydrochloride

Under ice-cooling, 20 ml of 4 M hydrochloric acid/ethyl acetate wasadded to 5.330 g (12.95 mmol) of the Boc-aminopropanol-ketoprofenobtained above, followed by stirring under ice-cooling for 15 minutesand at room temperature for 2 hours. After confirming disappearance ofBoc-aminopropanol-ketoprofen by TLC, the solvent was evaporated underreduced pressure, and the residue was subjected twice to decantationwith diethyl ether. Thereafter, the residue was dried under reducedpressure to quantitatively give the titled substance at a yield of 4.569g. The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.50 (5H, d, —CH(CH₃ )—), 2.08 (2H, m,—NHCH₂ CH₂ CH₂O—), 3.04 (2H, br, —NHCH₂ CH₂CH₂O—), 3.82 (1H, q,—OCOCH(CH₃)—), 4.16 (2H, m, —NHCH₂CH₂ CH₂ O—), 7.36-7.80 (9H, m,Aromatic H), 8.20 (br, H₃N⁺ CH₂—)

Example 2 Synthesis of Aminopropanol-Ketoprofen-Introduced SodiumHyaluronate

In 22.5 ml water/22.5 ml dioxane, 200 mg (0.5 mmol/disaccharide unit) ofsodium hyaluronate having a weight average molecular weight of 900,000was dissolved, and then 0.25 ml of 2 M aqueous hydroxysuccinimide (HOSu)solution, 0.25 ml of 1 mol/l aqueous WSCI.HCl solution and 0.5 ml of the0.5 M aqueous solution of aminopropanol-ketoprofen hydrochlorideobtained in Example 1 were added thereto in this order, followed bystirring overnight. To the reaction solution, 3 ml of 5% aqueous sodiumhydrogen carbonate solution was added, followed by stirring for 3 hoursand 20 minutes. After neutralizing the reaction solution by adding 86 μlof 50% acetic acid, 800 mg of sodium chloride was added thereto,followed by stirring. The mixture was precipitated by adding 200 ml ofethanol, and the precipitate was washed twice with 80% ethanol, twicewith ethanol and twice with diethyl ether and dried at room temperatureovernight under reduced pressure to give 198 mg portion of a whitesolid. The degree of substitution of ketoprofen was 15.5% by HPLCanalysis. The thus obtained substance was dissolved in PBS to aconcentration of 1.0% by weight to prepare a solution. The solution wasa colorless and transparent liquid, and the result of its filter passthrough test was “A”.

Example 3 Synthesis of Aminopropanol-Ketoprofen-Introduced SodiumHyaluronate

In 45 ml water/45 ml dioxane, 400 mg (1.0 mmol/disaccharide unit) ofhyaluronic acid having a weight average molecular weight of 900,000 wasdissolved, and then 1.66 mmol/1 ml water of HOSu, 0.83 mmol/1 ml waterof WSCI.HCl and 0.83 mmol/4 ml water of aminopropanol-ketoprofenhydrochloride obtained in Example 1 were added thereto in this order,followed by stirring overnight. To the reaction solution, 300 mg/l mlwater of sodium hydrogen carbonate was added, followed by stirring for 3hours and 10 minutes. After neutralizing the reaction solution by adding86 μl of acetic acid, 400 mg of sodium chloride was added thereto,followed by stirring. The mixture was precipitated by adding 300 ml ofethanol, and the precipitate was washed twice with 80% ethanol, twicewith ethanol and twice with diethyl ether and dried at room temperatureovernight under reduced pressure to give 246 mg of a white solid. Thedegree of substitution of ketoprofen was 26.3% by HPLC analysis. Thethus obtained substance was dissolved in PBS to a concentration of 1.0%by weight to prepare a solution. The solution was a colorless andtransparent liquid, and the result of its filter pass through test was“A”.

Example 4 Synthesis of Aminopropanol-Naproxen Hydrochloride 1) Synthesisof Boc-Aminopropanol-Naproxen

In 2 ml of dichloromethane, 350 mg (2 mmol) of Boc-aminopropanol and 462g (2 mmol) of naproxen (manufactured by Wako Pure Chemical Industries)were dissolved, and 48 mg (0.4 mmol) of DMAP and 422 g of WSCI.HCl (2.2mmol)/2 ml dichloromethane were added thereto in this order underice-cooling. After returning to room temperature and stirring for 4hours and 50 minutes, dichloromethane was evaporated under reducedpressure, and ethyl acetate was added thereto, followed by separation bywashing with 5% citric acid twice, water, 5% sodium hydrogen carbonatetwice, water and saturated brine consecutively. After dehydration dryingwith sodium sulfate, ethyl acetate was evaporated under reduced pressureto give 720 mg of white crystal of the titled compound (yield 93%). Thestructure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.42 (9H, s, Boc), 1.58 (3H, d,—OCOCH(CH₃ )—), 1.75 (2H, quant, —NHCH₂ CH₂ CH₂O—), 3.07 (2H, m, —NHCH₂CH₂CH₂O—), 3.85 (1H, q, —OCOCH(CH₃)—), 3.91 (3H, s, —OCH₃ ), 4.13 (2H,m, —NHCH₂CH₂ CH₂ O—), 4.63 (1H, br, —NHCH₂—), 7.09-7.75 (6H, m, AromaticH)

2) Synthesis of Aminopropanol-Naproxen Hydrochloride

In 1 ml of dichloromethane, 684 mg (1.76 mmol) of theBoc-aminopropanol-naproxen obtained above was dissolved, and 2 ml of 4 Mhydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring under ice-cooling for 20 minutes and at roomtemperature for 1 hour. After confirming disappearance ofBoc-aminopropanol-naproxen by TLC, and diethyl ether was added thereto,followed by decantation three times. Thereafter, the residue was driedunder reduced pressure to quantitatively give the titled substance at ayield of 564 mg. The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃+CD₃OD) δ (ppm)=1.57 (3H, d, —OCOCH(CH₃ )—), 2.02(2H, quant, —NHCH₂ CH₂ CH₂O—), 2.88 (2H, m, —NHCH₂ CH₂CH₂O—), 3.87 (1H,q, —OCOCH(CH₃)—), 3.90 (3H, s, —OCH₃ ), 4.17 (2H, m, —NHCH₂CH₂ CH₂ O—),7.08-7.73 (6H, m, Aromatic H), 8.10 (br, H₃N⁺ CH₂—)

Example 5 Synthesis of Aminopropanol-Naproxen-Introduced SodiumHyaluronate

In 11.5 ml water/11.5 ml dioxane, 100 mg (0.25 mmol/disaccharide unit)of sodium hyaluronate having a weight average molecular weight of900,000 was dissolved, and then HOSu (0.2 mmol)/0.1 ml water, WSCI.HCl(0.1 mmol)/0.1 ml water and aminopropanol-naproxen hydrochlorideobtained in Example 4 (0.1 mmol)/0.3 ml water were added thereto in thisorder, followed by stirring overnight. To the reaction solution, 1.5 mlof 5% aqueous sodium hydrogen carbonate solution was added, followed bystirring for 3 hours and 35 minutes. After neutralizing the reactionsolution by adding 43 μl of 50% acetic acid, 500 mg of sodium chloridewas added thereto, followed by stirring. The mixture was precipitated byadding 50 ml of ethanol, and the precipitate was washed twice with 80%ethanol, twice with ethanol and with diethyl ether and dried at roomtemperature overnight under reduced pressure to give 95 mg of a whitesolid. The degree of substitution of naproxen was 13.1% by HPLCanalysis. The thus obtained substance was dissolved in PBS to aconcentration of 1.0% by weight to prepare a solution. The solution wasa colorless and transparent liquid, and the result of its filter passthrough test was “A”.

Example 6 Synthesis of Aminopropanol-Ibuprofen Hydrochloride 1)Synthesis of Boc-Aminopropanol-Ibuprofen

In 2 ml of dichloromethane, 352 mg (2 mmol) of Boc-aminopropanol and 412g (2 mmol) of ibuprofen (manufactured by Wako Pure Chemical Industries)were dissolved, and 48 mg (0.4 mmol) of DMAP and 423 g (2.2 mmol) ofWSCI.HCl/2 ml dichloromethane were added thereto in this order underice-cooling. After returning to room temperature and stirring overnight,and ethyl acetate was added thereto, followed by separation by washingwith 5% citric acid twice, water, 5% sodium hydrogen carbonate twice,water and saturated brine consecutively. After dehydration drying withsodium sulfate, ethyl acetate was evaporated under reduced pressure togive 665 mg of the titled compound (yield 91%). The structure wasidentified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=0.88 (6H, d, —CH(CH₃) ₂), 1.44 (9H, s,Boc), 1.49 (3H, d, —OCOCH(CH₃ )—), 1.75 (2H, m, —NHCH₂ CH₂ CH₂O—), 1.85(1H, m, —CH₂ CH(CH₃)₂), 2.45 (2H, d, —CH₂ CH(CH₃)₂), 3.05 (2H, m, —NHCH₂CH₂CH₂O—), 3.69 (1H, q, —OCOCH(CH₃)—), 4.13 (2H, t, —NHCH₂CH₂ CH₂ O—),4.63 (1H, br, —NHCH₂—), 7.07-7.21 (4H, m, Aromatic H)

2) Synthesis of Aminopropanol-Ibuprofen Hydrochloride

In 1 ml of dichloromethane, 636 mg (1.75 mmol) of theBoc-aminopropanol-ibuprofen obtained above was dissolved, and 4 ml of 4M hydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring under ice-cooling for 10 minutes and at roomtemperature for 3 hours. After confirming disappearance ofBoc-aminopropanol-ibuprofen by TLC, diethyl ether was added thereto,followed by decantation three times. Thereafter, the residue was driedunder reduced pressure to give the titled substance at a yield of 406 mg(77%). The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=0.89 (6H, d, —CH(CH₃) ₂), 1.47 (3H, d,—OCOCH(CH₃ )—), 1.83 (1H, m, —CH₂ CH(CH₃)₂), 2.08 (2H, quant, —NHCH₂ CH₂CH₂O—), 2.44 (2H, d, —CH₂ CH(CH₃)₂), 3.01 (2H, t, —NHCH₂ CH₂CH₂O—), 3.71(1H, q, —OCOCH(CH₃)—), 4.11-4.27 (2H, m, —NHCH₂CH₂ CH₂ O—), 7.06-7.20(4H, m, Aromatic H), 8.25 (br, H₃N⁺ CH₂—)

Example 7 Synthesis of Aminopropanol-Ibuprofen-Introduced SodiumHyaluronate

In 11.5 ml water/11.5 ml dioxane, 100 mg (0.25 mmol/disaccharide unit)of sodium hyaluronate having a weight average molecular weight of900,000 was dissolved, and then HOSu (0.2 mmol)/0.1 ml water, WSCI.HCl(0.1 mmol)/0.1 ml water and the aminopropanol-ibuprofen hydrochlorideobtained in Example 6 (0.1 mmol)/0.3 ml water were added thereto in thisorder, followed by stirring overnight. To the reaction solution, 1.5 mlof 5% aqueous sodium hydrogen carbonate solution was added, followed bystirring for 3 hours and 35 minutes. After neutralizing the reactionsolution by adding 43 μl of 50% acetic acid, 500 mg of sodium chloridewas added thereto, followed by stirring. The mixture was precipitated byadding 50 ml of ethanol, and the precipitate was washed twice with 80%ethanol, twice with ethanol and with diethyl ether and dried at roomtemperature overnight under reduced pressure to give 93 mg of a whitesolid. The degree of substitution of ibuprofen was 16.4% by HPLCanalysis. The thus obtained substance was dissolved in PBS to aconcentration of 1.0% by weight to prepare a solution. The solution wasa colorless and transparent liquid, and the result of its filter passthrough test was “A”.

Example 8 Synthesis of Aminopropanol-Flurbiprofen Hydrochloride 1)Synthesis of Boc-Aminopropanol-Flurbiprofen

In 2 ml of dichloromethane, 352 mg (2 mmol) of Boc-aminopropanol and 489g (2 mmol) of flurbiprofen (manufactured by Wako Pure ChemicalIndustries) were dissolved, and 48 mg (0.4 mmol) of DMAP and 423 g (2.2mmol) of WSCI.HCl/2 ml dichloromethane were added thereto in this orderunder ice-cooling. After returning to room temperature and stirringovernight, ethyl acetate was added thereto, followed by separation bywashing with 5% citric acid twice, water, 5% sodium hydrogen carbonatetwice, water and saturated brine consecutively. After dehydration dryingwith sodium sulfate, ethyl acetate was evaporated under reduced pressureto give 753 mg of the titled compound (yield 94%). The structure wasidentified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.26 (9H, s, Boc), 1.54 (3H, d,—OCOCH(CH₃ )—), 1.80 (2H, quant, —NHCH₂ CH₂ CH₂O—), 3.13 (2H, m, —NHCH₂CH₂CH₂O—), 3.76 (1H, q, —OCOCH(CH₃)—), 4.15 (2H, m, —NHCH₂CH₂ CH₂ O—),4.66 (1H, br, —NHCH₂—), 7.10-7.55 (9H, m, Aromatic H)

2) Synthesis of Aminopropanol-Flurbiprofen Hydrochloride

In 1 ml of dichloromethane, 720 mg (1.79 mmol) of theBoc-aminopropanol-flurbiprofen obtained above was dissolved, and 4 ml of4 M hydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring under ice-cooling for 3 minutes and at roomtemperature for 3 hours and 10 minutes. After confirming disappearanceof Boc-aminopropanol-flurbiprofen by TLC, diethyl ether was addedthereto, followed by decantation twice. Thereafter, the residue wasdried under reduced pressure to give the titled substance at a yield of352 mg (94%). The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.51 (3H, d, —OCOCH(CH₃ )—), 2.10 (2H,quant, —NHCH₂ CH₂ CH₂O—), 3.05 (2H, t, —NHCH₂ CH₂CH₂O—), 3.76 (1H, q,—OCOCH(CH₃)—), 4.13-4.29 (2H, m, —NHCH₂CH₂ CH₂ O—), 7.07-7.53 (9H, m,Aromatic H), 8.27 (br, H₃N⁺ CH₂—)

Example 9 Synthesis of Aminopropanol-Flurbiprofen-Introduced SodiumHyaluronate

In 11.5 ml water/11.5 ml dioxane, 100 mg (0.25 mmol/disaccharide unit)of sodium hyaluronate having a weight average molecular weight of900,000 was dissolved, and then HOSu (0.2 mmol)/0.1 ml water, WSCI.HCl(0.1 mmol)/0.1 ml water and the aminopropanol-flurbiprofen hydrochlorideobtained in Example 8 (0.1 mmol)/0.3 ml water were added thereto in thisorder, followed by stirring overnight. To the reaction solution, 1.5 mlof 5% aqueous sodium hydrogen carbonate solution was added, followed bystirring for 3 hours and 35 minutes. After neutralizing the reactionsolution by adding 43 μl of 50% acetic acid, 500 mg of sodium chloridewas added thereto, followed by stifling. The mixture was precipitated byadding 50 ml of ethanol, and the precipitate was washed twice with 80%ethanol, twice with ethanol and with diethyl ether and dried at roomtemperature overnight under reduced pressure to give 94 mg of a whitesolid. The degree of substitution of flurbiprofen was 21.1% by HPLCanalysis. The thus obtained substance was dissolved in PBS to aconcentration of 1.0% by weight to prepare a solution. The solution wasa colorless and transparent liquid, and the result of its filter passthrough test was “A”.

Example 10 Synthesis of Aminopropanol-Acetylsalicylic AcidHydrochloride 1) Synthesis of Boc-Aminopropanol-Acetylsalicylic Acid

Boc-aminopropanol (2.11 mmol), acetylsalicylic acid (2.11 mmol)(manufactured by Wako Pure Chemical Industries) and DMAP (0.42 mmol)were dissolved in dichloromethane-dioxane (2:1, 6 ml), and WSCI.HCl(2.35 mmol) was added thereto under ice-cooling. After returning to roomtemperature and stifling overnight, ethyl acetate was added thereto,followed by separation by washing with 5% citric acid, 5% sodiumhydrogen carbonate and saturated brine consecutively. After dehydrationdrying with sodium sulfate, ethyl acetate was evaporated under reducedpressure. The thus obtained residue was purified by silica gel columnchromatography (hexane:ethyl acetate=3:1, 0.5% triethylamine) to givethe titled compound (298.0 mg, yield 48%). The structure was identifiedby ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.44 (9H, s, Boc), 1.90-1.96 (2H, m,BocHNCH₂ CH₂ CH₂O—), 2.35 (3H, s, —COCH₃ ), 3.24-3.28 (2H, m, BocHNCH₂CH₂CH₂O—), 4.35 (2H, t, BocHNCH₂CH₂ CH₂ O—), 4.78 (1H, s, NH), 7.11 (1H,dd, Aromatic), 7.32 (1H, td, Aromatic), 7.55-7.59 (1H, m, Aromatic),8.01 (1H, dd, Aromatic)

2) Synthesis of Aminopropanol-Acetylsalicylic Acid Hydrochloride

The Boc-aminopropanol-acetylsalicylic acid obtained above (0.814 mmol)was dissolved in dichloromethane (1 ml), and 4 N hydrochloric acid/ethylacetate (3 ml) was added thereto under ice-cooling, followed by stirringfor 2 hours. After confirming disappearance ofBoc-aminopropanol-acetylsalicylic acid by TLC, diethyl ether was addedthereto. The thus formed precipitate was centrifuged, and thesupernatant was subjected to decantation. The thus obtained precipitatewas dried under reduced pressure to give 213.9 mg (yield 96%) of thetitled compound. The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=2.22 (2H, t, H₂NCH₂ CH₂ CH₂O—), 2.35(3H, s, —COCH₃ ), 3.13 (2H, t, H₂NCH₂ CH₂CH₂O—), 4.41 (2H, t, H₂NCH₂CH₂CH₂ O—), 7.09 (1H, dd, Aromatic), 7.31 (1H, dt, Aromatic), 7.56 (1H, dt,Aromatic), 7.99 (1H, dd, Aromatic)

Example 11 Synthesis of Aminopropanol-Acetylsalicylic Acid-IntroducedSodium Hyaluronate

Hyaluronic acid (100 mg), 0.25 mmol/disaccharide unit having a weightaverage molecular weight of 900,000 was dissolved in water-dioxane(1:1), and 2 mol/L HOSu (0.1 ml), 1 mol/L WSCI.HCl (0.1 ml) and awater-dioxane (1:1) solution (2 ml) of the aminopropanol-acetylsalicylicacid hydrochloride obtained above in Example 10 were added thereto inthis order, followed by stirring overnight. To the reaction solution, 5%aqueous sodium hydrogen carbonate solution (1.5 ml) was added, followedby stirring for 3 hours. After neutralizing the reaction solution byadding 50% aqueous acetic acid solution (43 μl), sodium chloride (0.4 g)was added thereto, followed by stirring. The mixture was precipitated byadding ethanol (100 ml), and the precipitate was washed with 80% aqueousethanol solution, ethanol and diethyl ether, twice for each,consecutively. Thereafter, the precipitate was dried under reducedpressure to give the titled compound (97.7 mg). The degree ofsubstitution of acetylsalicylic acid was 13.5% when measured by anabsorptiometric method. The thus obtained substance was dissolved in PBSto a concentration of 1.0% by weight to prepare a solution. The solutionwas a colorless and transparent liquid, and the result of its filterpass through test was “A”.

Example 12 Synthesis of Aminopropanol-Felbinac Hydrochloride 1)Synthesis of Boc-Aminopropanol-Felbinac

Boc-aminopropanol (2.04 mmol), felbinac (2.04 mmol) (manufactured byAldrich Chem. Co.) and DMAP (0.41 mmol) were dissolved in dioxane (7 ml)and then a dioxane-dichloromethane (3:4) solution (7 ml) of WSCI.HCl(2.35 mmol) was added thereto under ice-cooling. The reaction solutionwas clarified by adding dimethylformamide (DMF) (3 ml) and then returnedto room temperature, followed by stirring overnight. Ethyl acetate wasadded thereto, followed by separation by washing with 5% aqueous citricacid solution, 5% aqueous sodium hydrogen carbonate solution andsaturated brine consecutively. After dehydration drying with sodiumsulfate, the solvent was evaporated under reduced pressure. The thusobtained residue was purified by silica gel column chromatography(hexane:ethyl acetate=3:1, 0.5% triethylamine) to give the titledcompound (623.0 mg, yield 83%). The structure was identified by ¹H-NMR(CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.44 (9H, s, Boc), 1.80-1.85 (2H, m,BocHNCH₂ CH₂ CH₂O—), 3.15-3.19 (2H, m, BocHNCH₂ CH₂CH₂O—), 3.67 (2H, s,PhCH₂ —), 4.18 (2H, t, BocHNCH₂CH₂ CH₂ O—), 4.67 (1H, s, NH), 7.34-7.59(9H, m, Aromatic)

2) Synthesis of Aminopropanol-Felbinac Hydrochloride

The Boc-aminopropanol-felbinac obtained above (1.69 mmol) was dissolvedin dichloromethane (1 ml), and 4 N hydrochloric acid/ethyl acetate (3ml) was added thereto under ice-cooling. The mixture was returned toroom temperature, followed by stirring for 2 hours. After confirmingdisappearance of Boc-aminopropanol-felbinac by TLC, diethyl ether wasadded thereto and the thus formed precipitate was centrifuged. The thusobtained precipitate was subjected to three times of decantation withdiethyl ether and then dried under reduced pressure to give the titledcompound (511.7 mg, yield 99%). The structure was identified by ¹H-NMR(CDCl₃:CD₃OD=1:1).

¹H-NMR (500 MHz, CDCl₃:CD₃OD=1:1) δ (ppm)=1.98-2.04 (2H, m, H₂NCH₂ CH₂CH₂O—), 2.95 (2H, t, H₂NCH₂ CH₂CH₂O—), 3.73 (2H, s, -PhCH₂ —), 4.23 (2H,t, H₂NCH₂CH₂ CH₂ O—), 7.33-7.59 (9H, m, Aromatic)

Example 13 Synthesis of Aminopropanol-Felbinac-Introduced HyaluronicAcid

Hyaluronic acid (200 mg) 0.5 mmol/disaccharide unit having a weightaverage molecular weight of 900,000 was dissolved in water-dioxane (1:1,45 ml), and 2 mol/L of HOSu (0.25 ml), 1 mol/L of WSCI.HCl (0.25 ml) andthe 0.5 M aqueous solution of felbinac propanolamine hydrochlorideobtained in Example 12 (0.5 ml) were added thereto in this order,followed by stirring overnight. To the reaction solution, 5% aqueoussodium hydrogen carbonate solution (3 ml) was added, followed bystirring for 3 hours. After neutralizing the reaction solution by adding50% aqueous acetic acid solution (86 μl), sodium chloride (0.8 g) wasadded thereto, followed by stirring. Ethanol (200 ml) was added thereto,followed by stirring. The thus formed precipitate was centrifuged, andthe resulting precipitate was washed with 80% aqueous ethanol solution,ethanol and diethyl ether consecutively, twice for each. The precipitatewas dried at room temperature overnight under reduced pressure to givethe titled compound (205.1 mg). The degree of substitution of felbinacwas 27.8% by HPLC analysis. The thus obtained substance was dissolved inPBS to a concentration of 1.0% by weight to prepare a solution. Thesolution was a colorless and transparent liquid, and the result of itsfilter pass through test was “A”.

Example 14 Synthesis of Aminopropanol-Fenbufen Hydrochloride 1)Synthesis of Boc-Aminopropanol-Fenbufen

Boc-aminopropanol (2.18 mmol), fenbufen (2.18 mmol) (manufactured by ICNBiochemicals Inc.) and DMAP (0.44 mmol) were dissolved inDMF-dichloromethane (5:3, 8 ml), and dichloromethane solution (5 ml) ofWSCI.HCl (2.48 mmol) was added thereto under ice-cooling. Aftergradually returning the reaction temperature to room temperature, themixture was stirred overnight. Ethyl acetate was added thereto, followedby separation by washing with 5% aqueous citric acid solution, 5%aqueous sodium hydrogen carbonate solution and saturated brineconsecutively. After dehydration drying with sodium sulfate, the solventwas evaporated under reduced pressure. The thus obtained residue waspurified by silica gel column chromatography (chloroform:ethylacetate=40:1, 0.5% triethylamine) to give the titled compound (747.8 mg,yield 83%). The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.44 (9H, s, Boc), 1.82-1.87 (2H, m,BocHNCH₂ CH₂ CH₂O—), 2.79 (2H, t, —COC₂H₄ CO—), 3.20-3.24 (2H, m,BocHNCH₂ CH₂CH₂O—), 3.36 (2H, t, —COC₂H₄ CO—) 4.19 (2H, t, BocHNCH₂CH₂CH₂ O—), 4.76 (1H, s, NH), 7.39-7.64 (5H, m, Aromatic), 7.70 (2H, td,Aromatic), 8.06 (2H, td, Aromatic)

2) Synthesis of Aminopropanol-Fenbufen Hydrochloride

The Boc-aminopropanol-fenbufen obtained above (1.82 mmol) was dissolvedin dichloromethane (4 ml), and under ice-cooling, 4 N hydrochloricacid.ethyl acetate solution (4 ml) was added thereto, and then themixture was gradually returned to room temperature and stirred for 90minutes. Precipitation of white precipitate was observed just after thecommencement of the reaction. After confirming disappearance ofBoc-aminopropanol-fenbufen by TLC, diethyl ether was added to thereaction solution, and the resulting white precipitate was centrifuged.The precipitate was washed three times with diethyl ether and then driedunder reduced pressure to give the titled compound (621.4 mg, yield98%). The structure was identified by ¹H-NMR (CDCl₃:CD₃OD=1:1).

¹H-NMR (500 MHz, CDCl₃:CD₃OD=1:1) δ (ppm)=2.01-2.07 (2H, m, H₂NCH₂ CH₂CH₂O—), 2.79 (2H, t, —COC₂H₄ CO—), 3.05 (2H, t, H₂NCH₂ CH₂CH₂O—), 3.44(2H, t, —COC₂H₄ CO—), 4.26 (2H, t, H₂NCH₂CH₂ CH₂ O—), 7.41-7.50 (3H, m,Aromatic), 7.66 (dd, 2H, Aromatic), 7.75 (d, 2H, Aromatic), 8.08 (d, 2H,Aromatic)

Example 15 Synthesis of Aminopropanol-Fenbufen-Introduced HyaluronicAcid

Hyaluronic acid (200 mg) 0.5 mmol/disaccharide unit having a weightaverage molecular weight of 900,000 was dissolved in water-dioxane (1:1,45 ml), and then 2 mol/L HOSu (0.25 ml), 1 mol/L WSCI.HCl (0.25 ml) anda water-dioxane (25:8) solution (0.66 ml) of the aminopropanol-fenbufenhydrochloride obtained in Example 14 were added thereto, followed bystirring overnight. To the reaction solution, 5% aqueous sodium hydrogencarbonate solution (3 ml) was added, followed by stirring for 3 hours.After neutralizing by adding 50% aqueous acetic acid solution (86 μl),sodium chloride (0.8 g) was added thereto, followed by stirring. Ethanol(200 ml) was added thereto, followed by stirring. The thus formedprecipitate was centrifuged, and the thus obtained precipitate waswashed with 80% aqueous ethanol solution, ethanol and diethyl ether,twice for each. The precipitate was dried under reduced pressure to givethe titled compound (214.1 mg). The degree of substitution of fenbufenwas 23.8% by HPLC analysis. The thus obtained substance was dissolved inPBS to a concentration of 1.0% by weight to prepare a solution. Thesolution was a colorless and transparent liquid, and the result of itsfilter pass through test was “A”.

Example 16 Synthesis of Aminopropanol-Mefenamic Acid Hydrochloride 1)Synthesis of Boc-Aminopropanol-Mefenamic Acid

Boc-aminopropanol (0.616 mmol), mefenamic acid (0.620 mmol)(manufactured by Wako Pure Chemical Industries) and DMAP (0.126 mmol)were dissolved in dichloromethane (3 ml), and a dichloromethane solution(1.5 ml) of WSCI.HCl (0.758 mmol) was added thereto, under ice-cooling.After gradually returning the reaction temperature to room temperature,the mixture was stirred overnight. The reaction solution was againice-cooled, and a dichloromethane solution (1 ml) of WSCI.HCl (0.207mmol) was added thereto, followed by stirring for 5 hours whilegradually returning to room temperature. Ethyl acetate was added to thereaction solution, and the mixture was washed with 5% aqueous citricacid solution, 5% aqueous sodium hydrogen carbonate solution andsaturated brine consecutively. After dehydration drying with sodiumsulfate, the solvent was evaporated under reduced pressure. The thusobtained residue was purified by silica gel column chromatography(hexane:ethyl acetate=6:1, 0.5% triethylamine) to give the titledcompound (190.4 mg, yield 78%). The structure was identified by ¹H-NMR(CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.45 (9H, s, Boc), 1.96-2.01 (2H, m,BocHNCH₂ CH₂ CH₂O—), 2.18 (3H, s, PhCH₃ ), 2.33 (3H, s, PhCH₃ ),3.31-3.32 (2H, m, BocHNCH₂ CH₂CH₂O—), 4.38 (2H, t, BocHNCH₂CH₂ CH₂ —),4.78 (1H, s, NH), 6.64-6.67 (1H, m, Aromatic), 6.74 (1H, dd, Aromatic),7.02-7.26 (4H, m, Aromatic), 7.94 (1H, dd, Aromatic), 9.24 (1H, s,-PhNHPh-)

2) Synthesis of Aminopropanol-Mefenamic Acid Hydrochloride

The Boc-aminopropanol-mefenamic acid obtained above (0.462 mmol) wasdissolved in dichloromethane (0.5 ml), and 4 N hydrochloric acid/ethylacetate (1.5 ml) was added thereto under ice-cooling, followed bystirring for 3 hours. After confirming disappearance ofBoc-aminopropanol-mefenamic acid by TLC, diethyl ether was added to thereaction solution, and the thus formed precipitate was centrifuged. Thethus obtained precipitate was washed with diethyl ether and then driedunder reduced pressure to give the titled compound (154.4 mg, qu.). Thestructure was identified by ¹H-NMR (CDCl₃). ¹H-NMR (500 MHz, CDCl₃) δ(ppm)=2.16 (3H, s, PhCH₃ ), 2.25-2.30 (2H, m, H₂NCH₂ CH₂ CH₂O—), 2.31(3H, s, PhCH₃ ), 3.20 (2H, t, H₂NCH₂ CH₂CH₂O—), 4.44 (2H, t, H₂NCH₂CH₂CH₂ O—), 6.63-6.66 (1H, m, Aromatic), 6.70-6.72 (1H, dd, Aromatic), 7.02(1H, d, Aromatic), 7.09 (1H, t, Aromatic), 7.14 (1H, d, Aromatic),7.22-7.25 (1H, m, Aromatic), 7.92 (1H, dd, Aromatic), 9.17 (1H, s,-PhNHPh-)

Example 17 Synthesis of Aminopropanol-Mefenamic Acid-IntroducedHyaluronic Acid

Hyaluronic acid (100 mg) 0.25 mmol/disaccharide unit having a weightaverage molecular weight of 900,000 was dissolved in water-dioxane (1:1,22.5 ml), and 2 mol/L HOSu (0.1 ml), 1 mol/L WSCI.HCl (0.1 ml) and awater-dioxane (1:1) solution (2 ml) of the aminopropanol-mefenamic acidhydrochloride (0.10 mmol) obtained in Example 16 were added thereto inthis order, followed by stirring overnight. To the reaction solution, 5%aqueous sodium hydrogen carbonate solution (1.5 ml) was added, followedby stirring for 4 hours. After neutralizing by adding 50% aqueous aceticacid solution (43 μl), sodium chloride (0.4 g) was added thereto,followed by stirring. Ethanol (100 ml) was added thereto, followed bystirring, and the thus formed precipitate was centrifuged. The thusobtained precipitate was washed with 80% aqueous ethanol solution,ethanol and diethyl ether, twice for each, consecutively. Theprecipitate was dried under reduced pressure to give the titled compound(101.7 mg). The degree of substitution of mefenamic acid was 17.5% whenmeasured by an absorptiometric method. The thus obtained substance wasdissolved in PBS to a concentration of 1.0% by weight to prepare asolution. The solution was a colorless and transparent liquid, and theresult of its filter pass through test was “A”.

Example 18 Synthesis of Aminopropanol-Diclofenac Hydrochloride 1)Synthesis of Boc-Aminopropanol-Diclofenac

In 1 ml of dichloromethane, 135.8 mg (0.775 mmol) of Boc-aminopropanolwas dissolved, 4 ml dichloromethane solution of 229.6 mg (0.775 mmol) ofdiclofenac (manufactured by Wako Pure Chemical Industries), 1 mldichloromethane solution of 18.9 mg (0.155 mmol) DMAP and 0.5 ml of DMFwere added thereto in this order, and 2 ml dichloromethane solution of191.4 mg (0.998 mmol) of WSCI.HCl was added thereto under ice-cooling,followed by stirring for 7 hours while gradually returning to roomtemperature. The reaction solution was again ice-cooled, and, as anadditional operation, 1 ml dichloromethane solution of 91.9 mg (0.310mmol) of diclofenac, 7.5 mg (0.061 mmol) of DMAP and 1 mldichloromethane solution of 70.9 mg (0.370 mmol) of WSCI.HCl were addedthereto in this order, followed by stirring while gradually returning toroom temperature. This additional operation was carried out 5 times.Ethyl acetate was added thereto, followed by separation by washing twicewith 5% aqueous citric acid solution, twice with 5% aqueous sodiumhydrogen carbonate solution and with saturated brine consecutively.After dehydration with sodium sulfate, ethyl acetate was evaporatedunder reduced pressure. The resulting residue was purified by silica gelcolumn chromatography to give 280.2 mg (80%) of the titled compound. Thestructure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.44 (9H, s, Boc), 1.85 (2H, quant,—NHCH₂ CH₂ CH₂O—), 3.16 (2H, q, —NHCH₂ CH₂CH₂O—), 3.82 (2H, s, Ph-CH₂—CO), 4.22 (2H, t, —NHCH₂CH₂ CH₂ O—), 4.68 (1H, s, NH), 6.54-7.35 (8H,m, Aromatic H, NH)

2) Synthesis of Aminopropanol-Diclofenac Hydrochloride

In 2 ml of dichloromethane, 1019 mg of the Boc-aminopropanol-diclofenacobtained above was dissolved, and 8 ml of 4 M hydrochloric acid/ethylacetate was added thereto under ice-cooling, followed by stirring for 3hours. After 150 ml of diethyl ether was added thereto forprecipitation, the precipitate was dried under reduced pressure. Thetitled compound was obtained at a yield of 791 mg (90%). The structurewas identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=2.13 (2H, quant, —NHCH₂ CH₂ CH₂O—), 3.08(2H, t, —NHCH₂ CH₂CH₂O—), 3.84 (2H, s, Ph-CH₂ —CO), 4.25 (2H, t,—NHCH₂CH₂ CH₂ O—), 6.52-7.33 (8H, m, Aromatic H, NH)

Example 19 Synthesis of Aminopropanol-Diclofenac-Introduced SodiumHyaluronate

In 56.3 ml water/56.3 ml dioxane, 500 mg (1.25 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 800,000was dissolved, and then HOSu (1 mmol)/0.5 ml water, WSCI.HCl (0.5mmol)/0.5 ml water and 0.5 mmol/(water:dioxane=1:1, 5 ml) of theaminopropanol-diclofenac hydrochloride obtained above in Example 18 wereadded thereto in this order, followed by stirring overnight. To thereaction solution, 7.5 ml of 5% aqueous sodium hydrogen carbonatesolution was added, followed by stirring for 3 hours and 40 minutes.After neutralizing the reaction solution by adding 215 μl of 50% aceticacid, 2.5 g of sodium chloride was added thereto, followed by stirring.The mixture was precipitated by adding 400 ml of ethanol, and theprecipitate was washed twice with 85% aqueous ethanol solution, twicewith ethanol and twice with diethyl ether and dried at room temperatureovernight under reduced pressure to give 541 mg of a white solid. Thedegree of substitution of diclofenac was 18.2% when measured with aspectrophotometer.

Example 20 Synthesis of Aminopropanol-Etodolac Hydrochloride 1)Synthesis of Boc-Aminopropanol-Etodolac

In 4 ml of dichloromethane, 178.8 mg (1.02 mmol) of Boc-aminopropanol,293.8 mg (1.02 mmol) of etodolac (manufactured by Wako Pure ChemicalIndustries) and 23.8 mg (0.20 mmol) of DMAP were dissolved, and 2 mldichloromethane solution of 233.8 mg (1.22 mmol) WSCI.HCl was addedthereto under ice-cooling, followed by stirring overnight whilegradually returning to room temperature. Further under ice-cooling, 2 mldichloromethane solution of 68.8 mg (0.36 mmol) WSCI.HCl was addedthereto, followed by stirring for 80 minutes while gradually returningto room temperature. Ethyl acetate was added thereto, followed byseparation by washing twice with 5% aqueous citric acid solution, twicewith 5% aqueous sodium hydrogen carbonate solution and with saturatedbrine consecutively. After dehydration drying with sodium sulfate, ethylacetate was evaporated under reduced pressure. The thus obtained residuewas purified by silica gel column chromatography to give 436.3 mg (96%)of the titled compound. The structure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=0.83 (3H, t, —CH₂ CH₃ ), 1.37 (3H, t,—CH₂ CH₃ ), 1.43 (9H, s, Boc), 1.79 (2H, quant, —NHCH₂ CH₂ CH₂O—), 3.14(2H, q, —NHCH₂ CH₂CH₂O—), 4.10-4.22 (2H, m, —NHCH₂CH₂ CH₂ O—), 4.63 (1H,s, NH), 7.00-7.37 (3H, m, Aromatic H), 8.97 (1H, s, NH)

2) Synthesis of Aminopropanol-Etodolac Hydrochloride

In 1 ml of dichloromethane, 421.5 mg (0.948 mmol) of theBoc-aminopropanol-etodolac obtained above was dissolved, and 3 ml of 4 Mhydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring for 3 hours. Diethyl ether and hexane were addedthereto for precipitation, and the precipitate was dried under reducedpressure. The precipitate was purified by silica gel columnchromatography to give 197.6 mg (55%) of the titled compound. Thestructure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=0.81 (3H, t, —CH₂ CH₃ ), 1.35 (3H, t,—CH₂ CH₃ ), 1.92-2.17 (4H, m, —CH₂ CH₃, —NHCH₂ CH₂ CH₂O—), 4.12 (1H,quant, —NHCH₂CH₂ CH₂ O—), 4.20 (1H, quant, —NHCH₂CH₂ CH₂ O—), 6.99-7.35(3H, m, Aromatic H), 8.99 (1H, s, NH)

Example 21 Synthesis of Aminopropanol-Etodolac-Introduced SodiumHyaluronate

In 12.8 ml water/12.8 ml dioxane, 114 mg (0.285 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 800,000was dissolved, and then 0.228 mmol HOSu/0.1 ml water, 0.114 mmolWSCI.HCl/0.1 ml water and 0.114 mmol/(2 ml water:dioxane=1:1) of theaminopropanol-etodolac hydrochloride obtained in Example 20 were addedthereto in this order, followed by stirring overnight. To the reactionsolution, 1.71 ml of 5% aqueous sodium hydrogen carbonate solution wasadded, followed by stirring for 4.5 hours. After neutralizing thereaction solution by adding 49 μl of 50% acetic acid, 456 mg of sodiumchloride was added thereto, followed by stirring. The mixture wasprecipitated by adding 110 ml of ethanol, and the precipitate was washedtwice with 80% ethanol, twice with ethanol and twice with diethyl etherand dried at room temperature overnight under reduced pressure to give111 mg of a white solid. The degree of substitution of etodolac was14.4% by HPLC analysis.

Example 22 Synthesis of Aminopropanol-Actarit Hydrochloride 1) Synthesisof Boc-Aminopropanol-Actarit

In 2 ml of dichloromethane, 123.1 mg (0.703 mmol) of theBoc-aminopropanol obtained in Reference Example 1 was dissolved, andthen a DMF solution (1 ml) of 136.0 mg (0.704 mmol) of actarit was addedthereto, and 17.1 mg (0.140 mmol) of DMAP and 175.4 mg (0.915 mmol) ofWSCI.HCl were added thereto in this order under ice-cooling, followed bystirring overnight while gradually returning to room temperature. Ethylacetate was added thereto, followed by separation by washing with 5%aqueous citric acid solution, 5% aqueous sodium hydrogen carbonatesolution and saturated brine consecutively. After dehydration dryingwith sodium sulfate, ethyl acetate was evaporated under reducedpressure. The precipitate was purified by silica gel columnchromatography to give 203.1 mg (83%) of the titled compound. Thestructure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.44 (9H, s, Boc), 1.80 (2H, quant,—NHCH₂ CH₂ CH₂O—), 2.18 (3H, s, NAc), 3.14 (2H, q, —NHCH₂ CH₂CH₂O—),3.59 (2H, s, Ph-CH₂ —CO), 4.15 (2H, t, —NHCH₂CH₂ CH₂ O—), 4.66 (1H, s,NH), 7.13 (1H, s, NH), 7.23 (2H, d, Aromatic H), 7.46 (2H, d, AromaticH)

In this connection, actarit was prepared by the following synthesismethod.

p-Aminophenylacetic acid (1.02 mmol) (manufactured by Wako Pure ChemicalIndustries) was dissolved in dichloromethane-methanol-water (1:3:1, 50ml), and acetic anhydride (2.12 mmol) was added thereto underice-cooling, followed by stirring overnight while gradually returning toroom temperature. The solvent was evaporated under reduced pressure togive the titled compound (196.4 mg, yield 99%). The structure wasidentified by ¹H-NMR.

¹H-NMR (500 MHz, CD₃OD) d (ppm)=2.11 (3H, s, Ac), 3.55 (2H, s, Ph-CH₂—), 7.21-7.49 (4H, m, Aromatic H)

2) Synthesis of Aminopropanol-Actarit Hydrochloride

In 2 ml of dichloromethane, 201.3 mg (0.574 mmol) of theBoc-aminopropanol-actarit obtained above was dissolved, and 3 ml of 4 Mhydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring for 3 hours. Diethyl ether was added thereto forprecipitation, and the precipitate was washed twice with diethyl etherand then dried under reduced pressure to give 161.3 mg (98%) of thetitled compound. The structure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CD₃OD) δ (ppm)=1.94-1.99 (2H, m, —NHCH₂ CH₂ CH₂O—),2.11 (3H, s, NAc), 2.94 (2H, t, —NHCH₂ CH₂CH₂O—), 3.63 (2H, s, Ph-CH₂—CO), 4.19 (2H, t, —NHCH₂CH₂ CH₂ O—), 7.22-7.51 (4H, m, Aromatic H)

Example 23 Synthesis of Aminopropanol-Actarit-Introduced SodiumHyaluronate

In 11.25 mL water/11.25 mL dioxane, 100 mg (0.25 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 800,000was dissolved, and then HOSu (0.2 mmol)/0.1 mL water, WSCI.HCl (0.1mmol)/0.1 mL water and the aminopropanol-actarit hydrochloride obtainedin Example 22 (0.1 mmol)/(water:dioxane=1:1, 2 mL) were added thereto inthis order, followed by stirring overnight. To the reaction solution,1.5 ml of 5% aqueous sodium hydrogen carbonate solution was added,followed by stirring for 3 hours. After neutralizing the reactionsolution by adding 43 of 50% aqueous acetic acid solution, 400 mg ofsodium chloride was added thereto, followed by stirring. The mixture wasprecipitated by adding 100 ml of ethanol, and the precipitate was washedtwice with 80% ethanol, twice with ethanol and with diethyl ether anddried at room temperature overnight under reduced pressure to give 97 mgof a white solid. The degree of substitution of actarit was 13.2% byHPLC analysis.

Example 24 Synthesis of Aminopropanol-Ketoprofen-Introduced SodiumHyaluronate

In 23 ml water/23 ml dioxane, 200 mg (0.5 mmol/disaccharide unit) ofhyaluronic acid having a weight average molecular weight of 900,000 wasdissolved, and 0.3 mmol/2 ml aqueous solution of HOSu, 0.15 mmol/2 mlaqueous solution of WSCI.HCl and 1.5 mmol/2 ml aqueous solution of theaminopropanol-ketoprofen hydrochloride obtained in Example 1 were addedthereto in this order, followed by stirring overnight. After 11.5 ml ofthe reaction solution was collected, 100 mg of sodium chloride was addedthereto, followed by stirring. The mixture was precipitated by adding 50ml of ethanol, and the precipitate was washed twice with 80% ethanol,twice with ethanol and twice with diethyl ether and dried at roomtemperature overnight under reduced pressure to give 35 mg of a whitepowder. The degree of substitution of ketoprofen was 7.2% by HPLCanalysis.

The thus obtained substance was dissolved in PBS to a concentration of1.0% by weight to prepare a solution. The solution was a colorless andtransparent liquid, and the result of its filter pass through test was“C”.

Reference Example 2 Synthesis of Boc-Serinol

Serinol (10.1 mmol) (manufactured by Aldrich Chem. Co.) was dissolved inwater-dioxane (1:1, 20 ml), and then a dioxane solution (15 ml) of Boc₂O(10.8 mmol) was added thereto under ice-cooling, followed by stirringovernight while returning to room temperature. The solvent wasevaporated under reduced pressure. The residue was washed with hexaneand then dried under reduced pressure to give the titled compound (1847mg, yield 95%). The structure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CD₃OD) δ (ppm)=1.44 (9H, s, Boc), 3.57-3.58 (5H, m,Serinol)

Example 25 Synthesis of Serinol-Ketoprofen Hydrochloride 1) Synthesis ofBoc-Serinol-Ketoprofen

Ketoprofen (1.11 mmol) (manufactured by Tokyo Kasei Kogyo) was dissolvedin dichloromethane (3 ml), and triethylamine (1.11 mmol) anddichloromethane solution (2 ml) of dimethylphosphinothioyl chloride(Mpt-Cl) (1.11 mmol) were added thereto in this order, followed bystirring for 25 minutes. Triethylamine (0.36 mmol) was further addedthereto, followed by stirring for 20 minutes. The reaction solution wasice-cooled, and triethylamine (1.11 mmol), DMAP (0.19 mmol) and theBoc-serinol obtained in Reference Example 2 (0.50 mmol) were addedthereto in this order, followed by stirring overnight by returning toroom temperature. The reaction solution was again ice-cooled, and 25%aqueous ammonia (2 ml) and dioxane (10 ml) were added thereto in thisorder, followed by stirring for 20 minutes. The reaction solution wasconcentrated to 5 ml, and ethyl acetate was added thereto. Separation bywashing with water, 5% aqueous citric acid solution, 5% aqueous sodiumhydrogen carbonate solution and saturated brine consecutively wascarried out, and after dehydration drying with sodium sulfate thesolvent was evaporated under reduced pressure. The precipitate waspurified by silica gel column chromatography (hexane:ethyl acetate=2:1,0.5% triethylamine) to give the titled compound (287.3 mg, yield 87%).The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.38-1.40 (9H, m, Boc), 1.51-1.53 (6H,m, —OCOCH(CH₃ )—), 3.76-3.81 (2H, m, —OCOCH(CH₃)—), 3.96-4.11 (4H, m,—CH₂ CH(NHBoc)CH₂ —), 4.61 (1H, btd, —CH₂ CH(NHBoc)CH₂—), 7.40-7.80(18H, m, Aromatic)

2) Synthesis of Serinol-Ketoprofen Hydrochloride

Boc-serinol-ketoprofen (0.428 mmol) was dissolved in dichloromethane (1ml), and 4 N hydrochloric acid/ethyl acetate (4 ml) was added theretounder ice-cooling, followed by stirring for 2 hours while graduallyreturning to room temperature. After confirming disappearance ofBoc-serinol-ketoprofen by TLC, diethyl ether and hexane were addedthereto, and the resulting precipitate was centrifuged. The thusobtained precipitate was dried under reduced pressure to give the titledcompound (243.6 mg, qu.). The structure was identified by ¹H-NMR(CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.49 (6H, t, —OCOCH(CH₃ )—), 3.79 (1H,m, —CH₂ CH(NHBoc)CH₂—), 4.00-4.53 (6H, m, Serinol, Ketoprofen),7.31-7.80 (18H, m, Aromatic)

Example 26 Synthesis of Serinol-Ketoprofen-Introduced Hyaluronic Acid

In water-dioxane (1:1, 22.5 ml), 0.25 mmol/disaccharide unit ofhyaluronic acid (100 mg) having a weight average molecular weight of900,000 was dissolved, and 2 mol/L HOSu (0.1 ml), 1 mol/L WSCI.HCl (0.1ml) and a dioxane solution (2 ml) of the serinol-ketoprofenhydrochloride obtained in Example 25 (0.10 mmol) were added thereto,followed by stirring overnight. To the reaction solution, 5% aqueoussodium hydrogen carbonate solution (1.5 ml) was added, followed bystirring for 4 hours. The mixture was neutralized by adding 50% aqueousacetic acid solution (43 μl), and sodium chloride (0.4 g) was addedthereto, followed by stirring. Ethanol (100 ml) was added thereto,followed by stirring, and the thus formed precipitate was centrifuged.The thus obtained precipitate was washed with 80% aqueous ethanolsolution, ethanol and diethyl ether consecutively, twice for each. Theprecipitate was dried under reduced pressure to give the titled compound(92.3 mg). The degree of substitution of ketoprofen was 11.2% by HPLCanalysis. The thus obtained substance was dissolved in PBS to aconcentration of 1.0% by weight to prepare a solution. The solution wasa colorless and transparent liquid, and the result of its filter passthrough test was “A”.

Example 27 Synthesis of 2-amino-1,5-pentanediol-ketoprofen hydrochloride

1) Synthesis of Boc-amino-1,5-pentanediol-ketoprofen

Boc-amino-1,5-pentanediol (Boc-NHCH(CH₂OH)CH₂CH₂CH₂OH, manufactured byAldrich Chem. Co.) (1.98 mmol) was dissolved in dichloromethane (2 ml),and a dichloromethane solution (4 ml) of ketoprofen (3.96 mmol)(manufactured by Tokyo Kasei Kogyo) and a dichloromethane solution (1ml) of DMAP (0.791 mmol) were added thereto in this order, followed bystirring. The reaction solution was ice-cooled, and a dichloromethanesolution (5 ml) of WSCI.HCl (4.93 mmol) was added thereto, followed bystirring overnight while gradually returning to room temperature. Thereaction solution was diluted with ethyl acetate, washed with 5% aqueouscitric acid solution, 5% aqueous sodium hydrogen carbonate solution andsaturated brine consecutively, followed by dehydration drying withsodium sulfate, and then the solvent was evaporated under reducedpressure. The thus obtained residue was purified by silica gel columnchromatography (hexane:ethyl acetate=5:2, 0.5% triethylamine) to givethe titled compound (1.361 g, yield 99%). The structure was identifiedby ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.39-1.40 (9H, m, Boc), 1.51-1.55 (6H,m, —OCOCH(CH₃ )—), 3.75-4.55 (8H, m, Ketoprofen,2-amino-1,5-pentanediol), 7.40-7.80 (18H, m, Aromatic)

2) Synthesis of 2-amino-1,5-pentanediol-ketoprofen hydrochloride

The Boc-amino-1,5-pentanediol-ketoprofen (1.95 mmol) obtained above wasdissolved in dichloromethane (1 ml), and 4 N hydrochloric acid/ethylacetate (4 ml) was added thereto under ice-cooling, followed by stirringfor 3 hours while gradually returning to room temperature. Hexane wasadded to the reaction solution, and the thus formed white precipitatewas centrifuged. The thus obtained precipitate was dried under reducedpressure to give the titled compound (1.20 g, yield 98%). The structurewas identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.50 (3H, d, —OCOCH(CH₃ )—), 1.51 (3H,d, —OCOCH(CH₃ )—), 3.47 (1H, bd, 2-amino-1,5-pentanediol), 3.44-4.48(6H, m, Ketoprofen, 2-amino-1,5-pentanediol), 7.33-7.84 (18H, m,Aromatic)

Example 28 Synthesis of 2-amino-1,5-pentanediol-ketoprofen-introducedhyaluronic acid

In water-dioxane (1:1, 30.8 ml), 0.34 mmol/disaccharide unit ofhyaluronic acid (137 mg) having a weight average molecular weight of900,000 was dissolved, and 2 mol/L HOSu (0.137 ml), 1 mol/L WSCI.HCl(0.137 ml) and a water-dioxane (1:1) solution (4 ml) of the2-amino-1,5-pentanediol-ketoprofen hydrochloride (0.137 mmol) obtainedin Example 27 were added thereto in this order, followed by stirringovernight. To the reaction solution, 5% aqueous sodium hydrogencarbonate solution (2.1 ml) was added, followed by stirring for 5 hours.After carrying out neutralization by adding 50% aqueous acetic acidsolution (59 μl), sodium chloride (0.548 g) was added thereto, followedby stirring. Ethanol (140 ml) was added thereto, followed by stirring,and the thus formed precipitate was centrifuged.

The thus obtained precipitate was washed with 80% aqueous ethanolsolution, ethanol and diethyl ether. The precipitate was dried underreduced pressure to give the titled compound (135.1 mg). The degree ofsubstitution of ketoprofen was 18.5% by HPLC analysis. The thus obtainedsubstance was dissolved in PBS to a concentration of 1.0% by weight toprepare a solution. The solution was a colorless and transparent liquid,and the result of its filter pass through test was “A”.

Example 29 Synthesis of 3-amino-1,2-propanediol-ketoprofenhydrochloride 1) Synthesis of Boc-amino-1,2-propanediol-ketoprofen

Boc-amino-1,2-propanediol (Boc-NHCH₂CH(OH)CH₂OH, manufactured by AldrichChem. Co.) (2.05 mmol) was dissolved in dichloromethane (2 ml), and adichloromethane solution (4 ml) of ketoprofen (4.11 mmol) (manufacturedby Tokyo Kasei Kogyo) and a dichloromethane solution (1 ml) of DMAP(0.803 mmol) were added thereto in this order, followed by stirring. Thereaction solution was ice-cooled, and a dichloromethane solution (5 ml)of WSCI.HCl (4.94 mmol) was added thereto, followed by stirringovernight while gradually returning to room temperature. The reactionsolution was ice-cooled, and a dichloromethane solution (1 ml) ofWSCI.HCl (1.24 mmol) was added thereto, followed by stirring at roomtemperature for 1 hour and then at 35° C. for 2 hours. Ethyl acetate wasadded thereto, followed by washing with 5% aqueous citric acid solution,5% aqueous sodium hydrogen carbonate solution and saturated brineconsecutively. Dehydration drying with sodium sulfate was carried out,and then the solvent was evaporated under reduced pressure. The thusobtained residue was purified by silica gel column chromatography(hexane:ethyl acetate=2:1, 0.5% triethylamine) to give the titledcompound (1.175 g, yield 87%). The structure was identified by ¹H-NMR(CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.36-1.40 (6H, m, Boc), 1.42-1.53 (6H,m, —OCOCH(CH₃ )—), 3.10-3.30 (2H, m, BocNHCH₂ —), 3.65-3.82 (2H, m,—OCOCH(CH₃)—), 3.99-4.36 (2H, m, BocNHCH₂(CH₂O—)CH₂ O—), 4.49-4.76 (1H,m, BocNH—), 5.04-5.09 (1H, m, BocNHCH₂(CHO—)CH₂O—), 7.38-7.80 (18H, m,Aromatic)

2) Synthesis of 3-amino-1,2-propanediol-ketoprofen hydrochloride

The Boc-amino-1,2-propanediol-ketoprofen (1.76 mmol) obtained above wasdissolved in dichloromethane (1 ml), and under ice-cooling, 4 Nhydrochloric acid/ethyl acetate (4 ml) was added thereto, followed bystirring for 3 hours. Hexane was added to the reaction solution, and thethus formed white precipitate was dried under reduced pressure to givethe titled compound (1.029 g, yield 97%). The structure was identifiedby ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.33-1.49 (6H, m, —OCOCH(CH₃ )—),3.02-3.20 (m, 2H, H₂NCH₂ (CH₂O—)CH₂O—), 3.56-3.82 (1H, m, —OCOCH(CH₃)—),3.90-4.15 (2H, m, H₂NCH₂(CHO—)CH₂ O—, —OCOCH(CH₃)—), 4.18-4.50 (1H, m,H₂NCH₂CH(O—)CH₂ O—), 5.35-5.37 (1H, m, H₂NCH₂ CH(O—)CH₂O—), 7.30-7.80(18H, m, Aromatic)

Example 30 Synthesis of 3-amino-1,2-propanediol-ketoprofen-introducedhyaluronic acid

In water-dioxane (1:1, 45 ml), hyaluronic acid (200 mg) 0.5mmol/disaccharide unit having a weight average molecular weight of900,000 was dissolved, and 2 mol/L HOSu (0.25 ml), 1 mol/L WSCI (0.25ml) and a water-dioxane (1:1) solution (4 ml) of the3-amino-1,2-propanediol-ketoprofen hydrochloride (0.20 mmol) obtained inExample 29 were added thereto in this order, followed by stirringovernight. To the reaction solution, 5% aqueous sodium hydrogencarbonate solution (3 ml) was added, followed by stirring for 4 hours.The mixture was neutralized by adding 50% aqueous acetic acid solution(86 μl), and sodium chloride (0.8 g) was added thereto, followed bystirring. Ethanol (200 ml) was added thereto, followed by stirring, andthe thus formed precipitate was centrifuged. The thus obtainedprecipitate was washed with 80% aqueous ethanol solution, ethanol anddiethyl ether. The precipitate was dried under reduced pressure to givethe titled compound (217.4 mg). The degree of substitution of ketoprofenwas 40.3% by HPLC analysis. The thus obtained substance was dissolved inPBS to a concentration of 1.0% by weight to prepare a solution. Thesolution was a colorless and transparent liquid, and the result of itsfilter pass through test was “A”.

Reference Example 3 Synthesis of Boc-Tris(Hydroxymethyl)Aminomethane

Tris(hydroxymethyl)aminomethane (10.1 mmol) was dissolved inwater-dioxane (1:2, 30 ml), and water-dioxane solution (1:9, 10 ml) ofBoc₂O (10.8 mmol) was added thereto, followed by stirring at roomtemperature for 45 minutes and then at 40° C. for 70 minutes. A dioxanesolution (3 ml) of Boc₂O (5.41 mmol) was added thereto, followed bystirring overnight while gradually returning to room temperature. Thesolvent was evaporated under reduced pressure. The precipitate waswashed with hexane and then dried under reduced pressure to give thetitled compound (2.21 g, yield 99%). The structure was identified by¹H-NMR.

¹H-NMR (500 MHz, CD₃OD) d (ppm)=1.44 (9H, s, Boc), 3.68 (6H, s, —C(CH₂OH)₃)

Example 31 Synthesis of Tris(Hydroxymethyl)Aminomethane-KetoprofenHydrochloride 1) Synthesis ofBoc-Tris(Hydroxymethyl)Aminomethane-Ketoprofen

In 3 ml of dichloromethane, 419 mg (1.65 mmol) of ketoprofen(manufactured by Tokyo Kasei Kogyo) was dissolved, and 230 μl (1.65mmol) of triethylamine and Mpt-Cl 213 mg (1.65 mmol)/2 mldichloromethane were added thereto in this order under ice-cooling,followed by stirring for 10 minutes. Next, 230 μl (1.65 mmol) oftriethylamine, mg (0.27 mmol) of DMAP and 110 mg (0.5 mmol) of theBoc-tris(hydroxymethyl)aminomethane (Boc-NHC(CH₂OH)₃) obtained inReference Example 3 were added thereto in this order, followed bystirring overnight after returning to room temperature. After adding 2ml of aqueous ammonia, dioxane was added thereto until dichloromethaneand aqueous ammonia became uniform, and the mixture was stirred for 40minutes. Dichloromethane was evaporated under reduced pressure, andethyl acetate was added to the residue, followed by separation bywashing twice with 5% citric acid, with water, twice with 5% sodiumhydrogen carbonate, with water and with saturated brine consecutively.After dehydration drying with sodium sulfate, ethyl acetate wasevaporated under reduced pressure and the residue was purified by silicagel column chromatography (dichloromethane:methanol=100:1→75:1). Thetitled compound was quantitatively obtained at a yield of 467 mg. Thestructure was identified by ¹H-NMR (CDCl₃) to confirm that 3 moleculesof ketoprofen were introduced into 1 molecule ofBoc-tris(hydroxymethyl)aminomethane.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.29 (9H, s, Boc), 1.44-1.54 (3H×3, m,—OCOCH(CH₃ )—), 3.76 (1H×3, q, —OCOCH(CH₃)—), 4.04-4.27 (6H, m, —NHC(CH₂O—KP)₃), 4.81 (1H, br, —NH—), 7.37-7.85 (9H×3, m, Aromatic H)

2) Synthesis of Tris(Hydroxymethyl)Aminomethane-Ketoprofen Hydrochloride

In 1 ml of dichloromethane, 453 mg (0.49 mmol) of theBoc-tris(hydroxymethyl)aminomethane-ketoprofen obtained above wasdissolved, and under ice-cooling, 3 ml of 4 M hydrochloric acid/ethylacetate was added thereto, followed by stirring for 30 minutes underice-cooling and then at room temperature for 1 hour and 30 minutes.After confirming disappearance ofBoc-tris(hydroxymethyl)aminomethane-ketoprofen by TLC, diethyl ether andhexane were added thereto for decantation. Thereafter, the precipitatewas dried under reduced pressure to give the titled compound with theyield of 411 mg (97%). The structure was identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.39-1.50 (3H×3, m, —OCOCH(CH₃ )—), 3.96(1H×3, q, —OCOCH(CH₃)—), 4.09-4.46 (6H, m, —NHC(CH₂ O—KP)₃), 7.25-7.80(9H×3, m, Aromatic H), 9.31 (br, H₃N⁺ CH₂—)

Example 32 Synthesis ofGlycine-Tris(Hydroxymethyl)Aminomethane-Ketoprofen Hydrochloride 1)Synthesis of Boc-Glycine-Tris(Hydroxymethyl)Aminomethane-Ketoprofen

In 1 ml of chloroform, 133 mg (0.76 mmol) of Boc-glycine was dissolved,and 106 μl (0.76 mmol) of triethylamine and Mpt-Cl 98 mg (0.76 mmol)/1ml chloroform were added thereto under ice-cooling, followed by stirringfor 10 minutes. Thereafter, 433 mg (0.5 mmol) of thetris(hydroxymethyl)aminomethane-ketoprofen hydrochloride obtained inExample 31/70 μl (0.5 mmol) of triethylamine/2 ml of chloroform, and 106μl (0.76 mmol) of triethylamine were gradually added thereto by dividinginto 4 portions. After stirring at room temperature for 1 hour, 106 μl(0.76 mmol) of triethylamine was further added thereto underice-cooling, and a mixed acid anhydride of Boc-glycine activated bydissolving 131 mg (0.75 mmol) of Boc-glycine in 1 ml chloroform and byadding, under ice-cooling, 105 μl (0.75 mmol) of triethylamine and 95 mg(0.75 mmol) Mpt-Cl/1 ml chloroform was added thereto, and the mixturewas stirred at room temperature overnight. Ethyl acetate was addedthereto, followed by separation by washing twice with 5% citric acid,water, twice with 5% sodium hydrogen carbonate, water and saturatedbrine consecutively. After dehydration drying with sodium sulfate, ethylacetate was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography (hexane:ethyl acetate=3:2)to give 411 mg of the titled compound (yield 55%). The structure wasidentified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.43 (9H, s, Boc), 1.45-1.52 (3H×3, m,—OCOCH(CH₃ )—), 3.56 (2H, br, —NHCH₂ CO—), 3.76 (1H×3, q, —OCOCH(CH₃)—),3.98-4.28 (6H, m, —NHC(CH₂ O—KP)₃), 5.51 (1H, br, —NHCH₂CO—), 6.63 (1H,br, —NHC(CH₂O—KP)₃), 7.34-7.83 (9H×3, m, Aromatic H)

2) Synthesis of Glycine-Tris(Hydroxymethyl)Aminomethane-KetoprofenHydrochloride

To 361 mg (0.37 mmol) of theBoc-glycine-tris(hydroxymethyl)aminomethane-ketoprofen obtained above, 2ml of 4 M hydrochloric acid/ethyl acetate was added under ice-coolingand stirred at room temperature for 2 hours. After confirmingdisappearance of Boc-glycine-tris(hydroxymethyl)aminomethane-ketoprofenby TLC, diethyl ether and hexane were added thereto for decantation.Thereafter, the precipitate was dried under reduced pressure to give thetitled compound quantitatively with the yield of 336 mg. The structurewas identified by ¹H-NMR (CDCl₃).

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.40 (3H×3, m, —OCOCH(CH₃ )—), 3.68-4.24(11H, m, 2H; —NHCH₂ CO—, 1H×3; —OCOCH(CH₃)—, 6H; —NHC(CH₂ O—KP)₃),7.27-7.82 (9H×3, m, Aromatic H), 8.31 (br, H₃N⁺ CH₂—)

Example 33 Synthesis ofGlycine-Tris(Hydroxymethyl)Aminomethane-Ketoprofen-Introduced HyaluronicAcid

In 11.5 ml water/11.5 ml dioxane, 100 mg (0.25 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 900,000was dissolved, and 2 mol/l HOSu/0.1 ml water, 1 mol/l WSCI.HCl/0.1 mlwater and 93 mg (0.1 mmol)/3 ml dioxane of theglycine-tris(hydroxymethyl)aminomethane-ketoprofen hydrochlorideobtained in Example 32 were added thereto in this order, followed bystirring overnight. To the reaction solution, 5% aqueous sodium hydrogencarbonate solution was added, followed by stirring for 4 hours and 45minutes. After neutralization of the reaction solution by adding 43μ of50% acetic acid, 400 mg of sodium chloride was added thereto, followedby stirring. The mixture was precipitated by adding 100 ml of ethanol,and the precipitate was washed twice with 80% ethanol, twice withethanol and with diethyl ether and dried at room temperature overnightunder reduced pressure to give 95 mg of white solid. The degree ofsubstitution of ketoprofen was 39% by HPLC analysis. The thus obtainedsubstance was dissolved in PBS to a concentration of 1.0% by weight toprepare a solution. The solution was a colorless and transparent liquid,and the result of its filter pass through test was “A”.

Reference Example 4 Synthesis of Boc-Aminopropyl Bromide

In 20 ml of dichloromethane, 1.222 g (5.58 mmol) of 3-bromopropylaminehydrobromide was dissolved, 0.778 ml (5.58 mmol) of triethylamine wasadded thereto under ice-cooling, and 50 ml dichloromethane solution of1.214 g (5.56 mmol) of Boc₂O was further added dropwise thereto in 10minutes, followed by stirring. After stirring at room temperature for 50minutes, ethyl acetate was added thereto, followed by separation bywashing with 5% aqueous citric acid solution, water and saturated brineconsecutively. After dehydration with sodium sulfate, the solvent wasevaporated under reduced pressure to give 1.304 of the titled compound(98%). The structure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.44 (9H, s, Boc), 2.05 (2H, quant,—NHCH₂ CH₂ CH₂Br), 3.28 (2H, q, —NHCH₂ CH₂CH₂Br), 3.44 (2H, t, —NHCH₂CH₂CH₂ Br), 4.64 (1H, s, NH)

Example 34 Synthesis of Aminopropanol-Diclofenac Hydrochloride (1)Boc-Aminopropanol-Diclofenac

In 3 ml of DMF, 1.476 g (4.64 mmol) of diclofenac sodium was dissolved,and 7 ml DMF solution of 1.105 g (4.64 mmol) of the Boc-aminopropylbromide obtained in Reference Example 6 was added dropwise thereto underice-cooling, followed by stirring overnight at room temperature and thenstirring at 60° C. for 10 hours. The mixture was stirred overnight atroom temperature and then stirred at 60° C. for 9 hours and furtherstirred at room temperature for 3 days. Ethyl acetate was added thereto,followed by separation by washing twice with 5% aqueous sodium hydrogencarbonate solution, water and saturated brine consecutively. Afterdehydration with sodium sulfate, ethyl acetate was evaporated underreduced pressure. The thus obtained residue was purified by silica gelcolumn chromatography (hexane:ethyl acetate=7:1, 0.5% triethylamine) togive 1.702 g (81%) of the titled compound.

(2) Aminopropanol-Diclofenac Hydrochloride

In 2 ml of dichloromethane, 1019 mg (2.25 mmol) of theBoc-aminopropanol-diclofenac obtained above was dissolved, and 8 ml of 4M hydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring for 3 hours. 150 ml of diethyl ether was addedthereto for precipitation, and the precipitate was dried under reducedpressure to give 791 mg (90%) of the titled compound. The structure wasidentified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=2.13 (2H, quant, —NHCH₂ CH₂ CH₂O—), 3.08(2H, t, —NHCH₂ CH₂CH₂O—), 3.84 (2H, s, Ph-CH₂ —CO), 4.25 (2H, t,—NHCH₂CH₂ CH₂ O—), 6.52-7.33 (8H, m, Aromatic H, NH)

Example 35 Synthesis of Aminopropanol-Diclofenac-Introduced SodiumHyaluronate (DS 4.3%)

In 57.5 mL water/57.5 mL dioxane, 500 mg (1.25 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 800,000was dissolved, and then 0.33 M HOSu/0.75 mL water, 0.16 M WSCI.HCl/0.75mL water and 0.16 M the aminopropanol-diclofenac hydrochloride obtainedabove in Example 34/0.75 mL water were added thereto in this order,followed by stirring overnight. Next, 375 mg of sodium hydrogencarbonate/3 ml water was added to the reaction solution, followed bystirring for 4 hours. After neutralization of the reaction solution byadding 108 μl of acetic acid, 3.0 g of sodium chloride was addedthereto, followed by stirring. The mixture was precipitated by adding200 ml of ethanol, and the precipitate was washed twice with 80%ethanol, twice with ethanol and twice with diethyl ether and dried atroom temperature overnight under reduced pressure to give 505 mg of awhite solid. The degree of substitution of diclofenac was 4.3% whenmeasured with a spectrophotometer.

Example 36 Synthesis of Aminopropanol-Diclofenac-Introduced SodiumHyaluronate (DS 9.7%)

In 57.5 ml water/57.5 ml dioxane, 500 mg (1.25 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 800,000was dissolved, and then 0.5 M HOSu/(water:dioxane=1:1) 1.0 ml, 0.25 MWSCI.HCl/(water:dioxane=1:1) 1.0 ml and the 0.25 Maminopropanol-diclofenac hydrochloride obtained above in Example34/(water:dioxane=1:1) 1.0 ml were added thereto in this order, followedby stirring overnight. Next, 380 mg of sodium hydrogen carbonate/5 mlwater was added to the reaction solution, followed by stirring for 4hours. After neutralization of the reaction solution by adding 108 μl ofacetic acid, 3.0 g of sodium chloride was added thereto, followed bystirring. The mixture was precipitated by adding 200 ml of ethanol, andthe precipitate was washed three times with 80% ethanol, twice withethanol and twice with diethyl ether and dried at room temperatureovernight under reduced pressure to give 503 mg of a white solid. Thedegree of substitution of diclofenac was 9.7% when measured with aspectrophotometer.

Example 37 Synthesis of Aminopropanol-Diclofenac-Introduced SodiumHyaluronate (65 kDa) Sodium (DS 17.1%) Using Hyaluronic Acid Having anAverage Molecular Weight of 65 kDa

In 22.5 mL water/22.5 mL dioxane, 200.8 mg (0.50 mmol/disaccharide unit)of hyaluronic acid having an average molecular weight of 65 kDa wasdissolved, and then 0.4 mL of 1 M HOSu, 0.4 mL of 0.5 M WSCI.HCl and 0.1M/(water:dioxane=1:1) 2.0 mL of the aminopropanol-diclofenachydrochloride obtained above in Example 34 were added thereto in thisorder, followed by stirring overnight. Next, 3 ml of 5% aqueous sodiumhydrogen carbonate solution was added to the reaction solution, followedby stirring for 3 hours. After neutralization of the reaction solutionby adding 86 μl of 50% acetic acid, 1.0 g of sodium chloride was addedthereto, followed by stirring. The mixture was precipitated by adding200 ml of ethanol, and the precipitate was washed twice with 85%ethanol, twice with ethanol and with diethyl ether and dried at roomtemperature overnight under reduced pressure to give 190.5 mg of a whitesolid. The degree of substitution of diclofenac was 17.1% when measuredwith a spectrophotometer.

Reference Example 5 boc-aminoethyl bromide

In 20 ml of dichloromethane, 2.155 g (10.5 mmol) of 3-bromoethylaminehydrobromide was dissolved, 1.463 ml (10.5 mmol) of triethylamine wasadded thereto under ice-cooling, and 5 ml dichloromethane solution of2.299 g (10.5 mmol) Boc₂O was further added thereto, followed bystirring. After stirring at room temperature for 90 minutes, ethylacetate was added thereto, followed by separation by washing with 5%aqueous citric acid solution, water and saturated brine consecutively.After dehydration with sodium sulfate, the solvent was evaporated underreduced pressure to give 2.287 g of the titled compound (97%). Thestructure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.45 (9H, s, Boc), 3.45-3.55 (4H, m,—NHCH₂CH₂ Br), 4.93 (1H, s, NH)

Example 38 Synthesis of Aminoethanol-Diclofenac Hydrochloride (1)Boc-Aminoethanol-Diclofenac

After ice-cooling 5 ml of DMF solution containing 2.287 g (10.2 mmol) ofthe Boc-aminoethyl bromide obtained in Reference Example 5, 6 ml of DMFsolution containing 3.255 g (10.2 mmol) of diclofenac sodium was addedthereto, followed by stirring at room temperature overnight. The mixturewas stirred at 60° C. for 11 hours and then stirred at room temperatureovernight. Ethyl acetate was added thereto, and separation by washingwith 5% aqueous sodium hydrogen carbonate solution, water and saturatedbrine consecutively was carried. After dehydration with sodium sulfate,ethyl acetate was evaporated under reduced pressure. The resultingresidue was purified by silica gel column chromatography (toluene:ethylacetate=20:1, 0.5% triethylamine) to give 2.675 g (60%) of the titledcompound. The structure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.42 (9H, s, Boc), 3.41 (2H, d, —NHCH₂CH₂O—), 3.83 (2H, s, Ph-CH₂ —CO), 4.21 (2H, t, —NHCH₂ CH₂ O—), 4.72 (1H,s, NH), 6.54-7.47 (8H, m, Aromatic H, NH)

(2) Aminoethanol-Diclofenac Hydrochloride

In 5 ml of dichloromethane, 2.108 g (4.80 mmol) of theBoc-aminoethanol-diclofenac obtained above was dissolved, and 20 ml of 4M hydrochloric acid/ethyl acetate was added thereto under ice-cooling,followed by stirring for 2.5 hours. Diethyl ether and hexane were addedthereto for precipitation, and the precipitate was dried under reducedpressure to give 1.775 g (98%) of the titled compound. The structure wasidentified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=3.18 (2H, t, NH₂ CH₂ CH₂O—), 3.94 (2H,s, Ph-CH₂ —CO), 4.37 (2H, t, NH₂CH₂ CH₂ O—), 6.47-7.31 (8H, m, AromaticH, NH)

Example 39 Synthesis of Aminoethanol-Diclofenac-Introduced SodiumHyaluronate (DS 14.7%)

In 57.5 mL water/57.5 mL dioxane, 500 mg (1.25 mmol/disaccharide unit)of hyaluronic acid having a weight average molecular weight of 800,000was dissolved, and then 0.5 mL of 2 M HOSu, 0.5 mL of 1 M WSCI.HCl and 3mL of a solution (water:dioxane=1:1) of 188.6 mg (0.5 mmol) of theaminoethanol-diclofenac hydrochloride obtained in Example 38 were addedthereto in this order, followed by stirring overnight. Next, 7.5 ml of5% aqueous sodium hydrogen carbonate solution was added thereto,followed by stirring for 4 hours. After neutralization of the reactionsolution by adding 215 μl of 50% acetic acid, 2.5 g of sodium chloridewas added thereto, followed by stirring. The mixture was precipitated byadding 500 ml of ethanol, and the precipitate was washed twice with 85%ethanol, twice with ethanol and twice with diethyl ether and dried atroom temperature overnight under reduced pressure to give 473.7 mg of awhite solid. The degree of substitution of diclofenac was 14.7% whenmeasured with a spectrophotometer.

Example 40 Synthesis of Diaminopropane-Diclofenac Hydrochloride (1)Boc-Propylamide-Diclofenac

In 3 ml of dichloromethane, 338.4 mg (1.94 mmol) of tert-butylN-(2-aminopropyl)carbamic acid (manufactured by Tokyo Kasei Kogyo) and694.4 mg (2.34 mmol) of diclofenac were dissolved, and underice-cooling, 59.0 mg (0.483 mmol) of DMAP and 505.3 mg (2.64 mmol) ofWSCI.HCl were added thereto, followed by stirring for 70 minutes andthen stirred at room temperature for 90 minutes. Ethyl acetate was addedthereto, followed by separation by washing with 5% aqueous citric acidsolution, 5% aqueous sodium hydrogen carbonate solution and saturatedbrine consecutively. After dehydration with sodium sulfate, ethylacetate was evaporated under reduced pressure. The resulting residue waspurified by silica gel column chromatography (hexane:ethyl acetate=2:1)to give 835.5 g (95%) of the titled compound. The structure wasidentified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.45 (9H, s, Boc), 1.60 (2H, quant,—NHCH₂ CH₂ CH₂NHBoc), 3.14 (2H, q, —NHCH₂CH₂ CH₂ NHBoc), 3.31 (2H, q,—NHCH₂ CH₂CH₂NHBoc), 3.69 (2H, s, Ph-CH₂ —CO), 4.93 (1H, s, NH),6.50-7.60 (9H, m, Aromatic H, NH)

(2) Diaminopropane-Diclofenac Hydrochloride

Under ice-cooling, 20 mL of 4 M hydrochloric acid/ethyl acetate wasadded to 1 mL dichloromethane solution of 825.0 mg (1.82 mmol) of theBoc-propylamide-diclofenac obtained above, followed by stirring for 2hours. Diethyl ether was added thereto for precipitation, and theprecipitate was dried under reduced pressure to give 714.5 mg (101%) ofthe titled compound. The structure was identified by ¹H-NMR.

¹H-NMR (500 MHz, CDCl₃) δ (ppm)=1.90 (2H, t, —NHCH₂ CH₂ CH₂NH₂), 2.99(2H, t, —NHCH₂CH₂ CH₂ NH₂), 3.26 (2H, d, —NHCH₂ CH₂CH₂NH₂), 3.71 (2H, s,Ph-CH₂ —CO), 6.40-7.49 (8H, m, Aromatic H, NH)

Example 41 Synthesis of Diaminopropane-Diclofenac-Introduced SodiumHyaluronate (DS 18.1%)

In 22.5 mL water/22.5 mL dioxane, 200 mg (0.5 mmol/disaccharide unit) ofhyaluronic acid having a weight average molecular weight of 800,000 wasdissolved, and then 0.2 mL of 2 M HOSu, 0.2 mL of 1 M WSCI.HCl and 1 mLof a solution (water:dioxane=1:1) of 78.4 mg (0.2 mmol) of thediaminopropane-diclofenac hydrochloride obtained in Example 40 wereadded thereto in this order, followed by stirring overnight. Next, 3 mlof 5% aqueous sodium hydrogen carbonate solution was added thereto,followed by stirring for 4 hours. After neutralization of the reactionsolution by adding 86 μl of 50% acetic acid, 1 g of sodium chloride wasadded thereto, followed by stirring. The mixture was precipitated byadding 200 ml of ethanol, and the precipitate was washed twice with 85%ethanol, twice with ethanol and with diethyl ether and dried at roomtemperature overnight under reduced pressure to give 206.2 mg of a whitesolid. The degree of substitution of diclofenac was 18.1% when measuredwith a spectrophotometer.

Example 42 Preparation of 1% Aminopropanol-Ketoprofen-Introduced SodiumHyaluronate Solution for Performance Test Use

To 22 mg of the aminopropanol-ketoprofen-introduced sodium hyaluronate(degree of substitution 26.3%) obtained in Example 3, 5 mM phosphatebuffered saline was added to give a total amount of 2.19 g, followed bystirring overnight to prepare a 1% solution. The solution was passedthrough a 0.45 μm filter and used as the titled solution. When theendotoxin content of this solution was measured by the endotoxin testmethod (colorimetric method) which is a general test method described inthe Pharmacopoeia of Japan, the endotoxin value was 0.0073 EU/M1.

Administration Test Example 43 Effect ofAminopropanol-Ketoprofen-Introduced Sodium Hyaluronate on theBradykinin-Induced Pain Model in Rats 1) Administration of TestSubstances

As General anesthesia, inhalation of isoflurane (Forane (registeredtrade mark), Dainippon Pharmaceutical Co., Ltd., concentration 3.0%,flow rate 2.0 liters/min) filled in a small animal anesthetizer (TK-4,manufactured by BioMachinery Co., Ltd.) was employed.

PBS, 1% sodium hyaluronate solution (HA), a 3.7 mg/ml ketoprofen sodiumsolution (KP) prepared by dissolving ketoprofen in PBS, and a 1% PBSsolution of the aminopropanol-ketoprofen-introduced sodium hyaluronate(KP-HA) prepared in Example 42 were used as the test substances.

Rats (Crj:SD(SPF), male, 7-weeks-old) were fixed in the supine positionunder ether anesthesia, and a wide area around the knee joint of theleft hind paw was shaved with an electric clipper. After disinfectingthe area around the joint by spraying 70% alcohol, the above-describedtest substances were administered into the knee joint cavity of the lefthind paw at a dose of 20 μl/joint using a 29G needle-tipped syringe forinsulin use (manufactured by Terumo Corp.). This procedure was performedusing 5 cases (n=5) for each test substance group.

2) Administration of Algesic Substance (BK+PGE₂ Solution)

After 1 day of the administration of each test substance, rats werefixed in the supine position under no anesthesia. After disinfecting thearea around the joint by spraying 70% alcohol, a mixed solution ofbradykinin (BK) and prostaglandin E₂ (PGE₂) as algesic substances wasadministered into the knee joint cavity of the left hind paw at a doseof 50 μl/joint using a 29G needle-tipped syringe for insulin use(manufactured by Terumo Corp., thickness of the needle is 0.33 mm).Additionally, this algesic substance solution was produced in such amanner that the final concentrations of BK and PGE₂ became 4 μg/ml and 2μg/ml, respectively. Pain reactions were visually observed right afterthe administration of the algesic agent.

3) Pain Observation

For about 2 minutes after administration of the algesic substances, thebehavioral manifestations in gait such as “lifting the foot”, “walkingon three legs” and “claudication” were visually observed and scored. Thepain scores were assigned as lifting the foot: 1 point addition andclaudication or walking on three legs: 1 point addition, and evaluatedby a stage of from 0 to 2 points. In addition, the evaluation wasperformed under blinded conditions. A graph in which pain reactions ofrespective individuals were scored is shown in FIG. 1.

In FIG. 1, the results are shown by average pain score±standarddeviation.

Consequently, the pain suppressing effects were observed in order ofKP-HA>KP>HA, in comparison with the PBS administration group.

Example 44 Effects of Intra-Articular Injection ofAminopropanol-Ketoprofen-Introduced Sodium Hyaluronate on the 1% SilverNitrate-Induced Pain Model in Rats 1) Administration of Pain InducingSubstances

As General anesthesia, inhalation of isoflurane (Forane (registeredtrade mark), Dainippon Pharmaceutical Co., Ltd., concentration 3.0%,flow rate 2.0 liters/min) filled in a small animal anesthetizer (TK-4,manufactured by BioMachinery Co., Ltd.) was employed.

Rats (Crj: SD (SPF), male, 6-weeks-old) were fixed in the supineposition under ether anesthesia, and a wide area around the knee jointof the left hind paw was shaved with an electric clipper. Afterdisinfecting the area around the joint by spraying 70% alcohol, 1%silver nitrate solution was administered into the knee joint cavity ofthe left hind paw at a dose of 50 μl/joint using a 29G needle-tippedsyringe for insulin use (manufactured by Terumo Corp.).

2) Administration of Test Substances

As the test substances, 1% sodium hyaluronate solution (HA) and 1%solution of the aminopropanol-ketoprofen-introduced sodium hyaluronate(KP-HA) prepared in Example 42, each using PBS as the solvent, wereprepared. Rats were divided into 2 groups, each including 5 animals, andeach test substance was administered to respective groups on 24 hoursafter the administration of 1% silver nitrate solution. Regarding theadministration method, as in the case of algesic substances, the areaaround the joint was disinfected by spraying 70% alcohol underinhalation anesthesia by isoflurane, and each test substance wasadministered into the knee joint cavity of the left hind paw at a doseof 40 μl/joint using a 29G needle-tipped syringe for insulin use (n=5).

3) Evaluation Method

The walking of each group was visually observed and scored by using thefollowing pain score table which was figured walking in score underblinded conditions. The results are shown in FIG. 2.

In FIG. 2, the results are shown by average pain score±standarddeviation.

Score 0: Normal (includes nearly normal)

-   -   1: Mild claudication    -   2: Severe claudication    -   3: Walking on three legs

In addition, the weight loading on the silver nitrate-injected paw (lefthind paw) was measured by using a weighting activity analgesia meter(manufactured by Tokken Inc.), and the weight loading rate wascalculated by dividing the measured value by the body weight.(Incidentally, weight loading rate was about 32% at the normal animals.)The measurement was performed once a day until 2 days after theadministration of each test substance. The results are shown in FIG. 3.

As shown in FIG. 2, the pain score was gradually reduced in both of theHA administration group and KP-HA administration group, but the degreeof pain relief (degree of recovery from pain) was quicker in the KP-HAadministration group than the HA administration group. In addition, theweight loading rate generally becomes high as the recovery from painprogresses on the measurement of the rate, but as shown in FIG. 3, theweight loading rate became significantly higher within a short period oftime in animals given KP-HA compared to those given HA. Relationshipbetween the KP-HA group and the HA group in the results of FIG. 2 andFIG. 3 was the same.

Example 45 Examination on the Sustained Release Property ofNSAIDs-Introduced Hyaluronic Acid in the Rabbit Knee Joint 1)Administration Method of Test Substances

The 1% aminopropanol-ketoprofen-introduced sodium hyaluronate solution(KP-HA) obtained in Example 42, a ketoprofen solution (KP) in which 1.42mg of ketoprofen was dissolved in 1 ml of PBS, and a mixed solution ofketoprofen and HA (KP+HA) in which 1.41 mg of ketoprofen was dissolvedin 1 ml of 1% HA solution were used as the test substances.

Using five rabbits for each test substance, rabbits were fixed in thesupine position under ketamine general anesthesia (1 ml/head, i.v.), anda wide area around the knee joint of the left hind paw was shaved withan electric clipper. Each of the above-described test substances wasadministered into the joint cavity from outside of the rabbit knee at adose of 300 μl using a 1 ml syringe equipped with a 25G needle(manufactured by Terumo Corp., thickness of the needle is 0.5 mm).

Autopsy was performed on 6, 12, 24 hours and 2, 4 days afteradministration of test substances.

2) Measuring Method of the Amounts of Free Type KP and Binding Type KPin Synovial Fluid

The rabbits were sacrificed by exsanguination under ketamine generalanesthesia. After all synovial fluid was collected, the joint cavity waswashed 2 times with 2 mL saline into the joint cavity of the dissectedknee using a 25G needle. The wash fluids were also collected. Amounts ofKP and HA-KP in the synovial fluid combined with the recovered washfluids were measured by the following procedure.

By adding 1 N HCl (0.2 ml) to the synovial fluid (4 ml vol.),hydrochloric acid acidity was confirmed, and then ethyl acetate havingthe same volume of the solution was added and vigorously stirred and theupper organic layer was recovered. This extraction operation wasperformed 3 times in total. An acetonitrile solution was added to therecovered organic layer to make it into an acetonitrile solution, andthe amount of free KP was measured by using HPLC (Amount of free type KPin synovial fluid).

Next, the water layer obtained by the above-described extractionoperation was adjusted to strongly basic state by adding 1 N NaOH andstirred at room temperature for 1 hour. Subsequently, the water layerwas ice-cooled, adjusted to hydrochloric acid acidic state by slowlyadding 4 N HCl while stirring, and then vigorously stirred by addingethyl acetate having the same volume of the solution to recover theupper organic layer. This extraction operation was performed 3 times intotal. An acetonitrile solution was added to the recovered organic layerto make it into an acetonitrile solution, and the amount of HA-KP(amount of binding type KP) was measured by using HPLC (Amount of boundHA-KP in synovial fluid).

3) Measuring Method of the Amount of KP in the Digestive Fluid ofSynovium

Synovium was separated and collected from the knee joint after recoveryof the synovial fluid of the above-described (2). The collected synoviumwas thoroughly washed with 100 ml of saline to completely remove theadherent synovial fluid. After removing the patella, the synovium wasput into a tube, 5 ml of proteinase K (Lot No. 102K8633, manufactured bySIGMA) prepared to be a concentration of 2 mg/ml with 10 mM sodiumacetate solution (pH 7.5) was added thereto, and enzyme digestion wasperformed at 55° C. for 41 hours while optionally stirring using Vortex.After the digestion, the enzyme was deactivated by incubating at 100° C.for 5 minutes, and the amount of KP in the thus obtained digestive fluidwas measured by the following procedure.

A 1/4 volume of 4 N NaOH was added to the thus obtained digestive fluidand stirred at room temperature for 1 hour. Subsequently, the solutionwas ice-cooled, adjusted to hydrochloric acid acidic state by slowlyadding 4 N HCl, and then vigorously stirred by adding diethyl etherhaving the same volume of the solution to remove the upper organiclayer. This degreasing operation was performed 3 times in total. Underice-cooling, 4 N HCl was added to the solution after the degreasingoperation and stirred to confirm hydrochloric acid acidic state and thenvigorously stirred by adding ethyl acetate having the same volume of thesolution to recover the upper organic layer. This extraction operationwas performed 3 times in total. An acetonitrile solution was added tothe thus recovered organic layer to make it into an acetonitrilesolution, and the amount of free KP was measured by using HPLC.

Amount of Free Type KP in Synovium Digestive Fluid:

Residual ratios of KP and HA-KP in the synovial fluid and synoviumdigestive fluid were calculated with time. (Table 1, FIG. 4)

TABLE 1 Test 6 hours 12 hours 24 hours 2 days 4 days substance SampleExisting form (%) (%) (%) (%) (%) KP synovial fluid Free type KP 0(single drug) synovium 0 KP + HA synovial fluid Free type KP 0.07 0(mixture) synovium 0 0 KP-HA synovial fluid Free type KP 0.13 0.14 0.220.16 0 (conjugate) Binding type KP 46.56 34.94 18.95 8.11 0.63 synovium11.3 5.20 32.90 12.10 7.40

When KP (single drug) or KP+HA (mixture) was administered as the testsubstance, KP disappeared from the synovial fluid and synovium within 6hours and 12 hours. However, when the KP-HA (conjugate) as a substanceof the present invention was administered, the presence of KP wasconfirmed in both of the synovial fluid and synovium even after 4 days,so that it was considered that KP shows its long-lasting effect bypersistently presenting in the administered site.

It is presumed that the joint pain is generated not via a cartilagewhich is an neurogenic tissue but via a synovium, and in addition, it isconsidered that when NSAIDs are administered into a joint cavity, NSAIDsare penetrated into a synovium rapidly and exert the effect bytransferring to the synovium. Thus, it is considered that keeping ofNSAIDs concentration in the synovium is greatly concerned in thelong-lasting analgesic effect and sustained release effect. As shown inthe above table, when KP (single drug) was administered, it disappearedfrom the synovium within 6 hours by its passing through the synovium andmetabolism. However, when KP-HA (conjugate) was administered, KP waspersistently maintained also in the synovium, thus indicating that thisis more effective as sustained release preparations of NSAIDs.

Example 46 Effects of Intra-Articular Injection ofAminopropanol-Diclofenac-Introduced Sodium Hyaluronate Having DifferentDegree of Substitution (DS) on the 1% Silver Nitrate Induced Pain Modelin Rats

Evaluation of the intra-articular injection of the following testsubstances was performed in accordance with the procedure in the aboveExample 44.

Test Substances:

(i) PBS solution of 1% aminopropanol-diclofenac-introduced sodiumhyaluronate (DS 18.2%) obtained in Example 19(ii) PBS solution of 1% aminopropanol-diclofenac-introduced sodiumhyaluronate (DS 9.7%) obtained in Example 36(iii) PBS solution of 1% aminopropanol-diclofenac-introduced sodiumhyaluronate (DS 4.3%) obtained in Example 35

(iv) PBS

In the same manner as in the above Example 44, the walking of each groupwas visually observed and scored by using the pain score table which wasfigures walking in score under blinded conditions. The results are shownin FIG. 5. The results are shown by average pain score±standarddeviation, and the DS 18%, DS 10% and DS 4% respectively correspond toDS 18.2%, DS 9.7% and DS 4.3%. In addition, in FIG. 5, * indicates thatthere is a significant difference against PBS with a level ofsignificance of 0.01<p<0.05, and ** indicates that there is asignificant difference against PBS with a level of significance ofp<0.01.

Consequently, all of the diclofenac-introduced sodium hyaluronatederivatives of DS 18.2%, DS 9.7% and DS 4.3% as the test substancesshowed analgesic effect. Particularly, the test substances of DS 18.2%and DS 9.7% showed a remarkable analgesic effect in comparison with PBS.

In addition, the analgesic effect was improved dependently on theincrease of the degree of substitution (DS) of diclofenac.

Reference Example 6 Effects of Oral Administration of Diclofenac Sodiumon the 1% Silver Nitrate Induced Pain Model in Rats

The test was performed in accordance with the procedure in the aboveExample 44, and the following test substances was orally administeredand evaluated. Test substances were orally administered by using a sondefor oral administration to rat (manufactured by Fuchigami Kikai) at adose of 1 ml/head.

Test Substances:

(i) 1% diclofenac sodium suspension (10% gum arabic)(ii) 0.02% diclofenac sodium suspension (10% gum arabic)

Additionally, the (i) (high dose group) corresponds to theadministration of 50 mg/kg as diclofenac sodium, and the (ii) (low dosegroup) corresponds to the administration of 1 mg/kg as diclofenac sodiumwhich is almost the same amount of the clinical dose.

In the same manner as in the above Example 46, the walking of each groupwas visually observed and scored by using the pain score table which wasfigured walking in score under blinded conditions. The results are shownin FIG. 6. In FIG. 6, the “Diclofenac Na (p.o.) 50 mg/kg” corresponds tothe above-described (i) (high dose group), and the “Diclofenac Na (p.o.)1 mg/kg” corresponds to the above-described (ii) (low dose group).Additionally, the results of pain scores by intra-articular injection ofthe diclofenac-introduced hyaluronic acid derivative (DS 18.2%) and PBS,measured in the above Example 46, were also described as references inFIG. 6 as “Diclofenac-HA” and “PBS”, respectively.

Consequently, oral administration of the high dose (50 mg/kg) ofdiclofenac sodium showed analgesic effect, however positive fecal occultblood reaction, jaundice-like symptom and body weight loss were observedfrom the next day. The dose of the high dose group is scores of timeslarger than the clinical dose, and is not a practical dose in terms ofadverse effects and toxicity.

In the low dose group (1 mg/kg) with oral administration, which isalmost the clinical dose, the effect was not found in comparison withthe PBS intra-articular injection group.

On the other hand, intra-articular injection of thediclofenac-introduced hyaluronic acid derivative showed the analgesiceffect after the administration. Additionally, the adverse effect causedby oral administration of the high dose (50 mg/kg) of diclofenac sodiumwas not observed, so that its high availability was confirmed.

Reference Example 7 Effects of Intra-Articular Injection of DiclofenacSingle Drug and Hyaluronic Acid on the 1% Silver Nitrate Induced PainModel in Rats

The test was performed in accordance with the procedure in the aboveExample 44, and the following test substances were intra-articulatelyadministered and evaluated.

Test Substances:

(i) 0.1% diclofenac solution(ii) 0.1% diclofenac/1% hyaluronic acid mixed solution(iii) PBS

In the same manner as in the above Example 46, the walking of each group(n=9) was visually observed and scored using the pain score table whichwas figured walking in score under blinded conditions. The results areshown in FIG. 7. In FIG. 7, the results are shown by average painscore±standard deviation, and the “0.1% Diclofenac Na” corresponds tothe above-described (i) 0.1% Diclofenac solution, and the “0.1%Diclo+HA” to the above-described 0.1% diclofenac/1% hyaluronic acidmixed solution. Additionally, the results of the diclofenac-introducedhyaluronic acid derivative (DS 18.2%) measured in the above Example 46were also described as references in FIG. 7 as “Dic-HA (conjugate)”.

Consequently, the diclofenac single drug and the mixture of diclofenacand hyaluronic acid did not show significant effect in comparison withPBS as the control group.

Example 47 Effects of Intra-Articular Injection ofAminopropanol-Diclofenac-Introduced Sodium (65 kDa) Hyaluronate,Diaminopropane-Diclofenac-Introduced Sodium Hyaluronate andAminoethanol-Diclofenac-Introduced Sodium Hyaluronate on the 1% SilverNitrate Induced Pain Model in Rats

The test was performed in accordance with the procedure in the aboveExample 44, and the following test substances were intra-articulatelyadministered and evaluated.

Test Substances:

(i) PBS solution of the 1% aminopropanol-diclofenac-introduced sodium(65 kDa) hyaluronate obtained in Example 37(ii) PBS solution of the 1% diaminopropane-diclofenac-introduced sodiumhyaluronate obtained in Example 41(iii) PBS solution of the 1% aminoethanol-diclofenac-introduced sodiumhyaluronate obtained in Example 39

(iv) PBS

In the same manner as in the above Example 44, the walking of each group(n=7) was visually observed and scores by using the pain score tablewhich was figured walking in score under blinded conditions. The resultsare shown in FIG. 8. In FIG. 8, the results are shown by average painscore, and the “65 kDa” corresponds to the above-described (i) PBSsolution of the 1% aminopropanol-diclofenac-introduced sodium (65 kDa)hyaluronate, and the “diamide” to the above-described (ii) PBS solutionof the 1% diaminopropane-diclofenac-introduced sodium hyaluronate andthe “C2” to the above-described (iii) PBS solution of the 1%aminoethanol-diclofenac-introduced sodium hyaluronate. In addition, inFIG. 8, Δ indicates that there is a significant difference against PBSwith a level of significance of 0.05<p<0.1, * indicates that there is asignificant difference against PBS with a level of significance of0.01<p<0.05, and ** indicates that there is a significant differenceagainst PBS with a level of significance of p<0.01.

Consequently, the aminoethanol-diclofenac-introduced sodium hyaluronatesolution which used aminoethanol, wherein the number of spacer carbonsis smaller than aminopropanol by a factor of 1, also showed remarkableanalgesic effect. However, the diaminopropane-diclofenac-introducedsodium hyaluronate solution, in which diclofenac was introduced throughan amide bond, had no effect on the test system of this example. Inaddition, the diclofenac-introduced hyaluronic acid derivative, whichused a hyaluronic acid having a molecular weight of 65 kDa, showeddiminished analgesic effect in comparison with the case of the molecularweight of 800,000.

These results showed that the analgesic effect depends on the bindingform with diclofenac or the molecular weight of hyaluronic acid.

Example 48 Effects of Diclofenac Sodium Single Drug andDiclofenac-Introduced Hyaluronic Acid Derivative on COX-2 (In Vitro)

The COX-2 inhibitory activity of the following test substances wasevaluated using Chemiluminescent COX Inhibitor Screening Assay Kit(Cayman) (a kit for screening inhibitors using the peroxidase activityof sheep-derived COX-2 as the index).

Test Substances:

(i) aminopropanol-diclofenac-introduced sodium hyaluronate aqueoussolution obtained in Example 19 (Dic-C3-HA) (corresponds to 200 μg/mlHA, corresponds to 80 μM Diclofenac)(ii) aminoethanol-diclofenac-introduced sodium hyaluronate aqueoussolution obtained by the same procedure of Example 39 (Dic-C2-HA, DS18.5%) (corresponds to 200 μg/ml HA, corresponds to 80 μM Diclofenac)(iii) 80 μM diclofenac sodium aqueous solution(iv) 200 μg/ml sodium hyaluronate (HA) aqueous solution

By preparing stock solutions of the above-described various testsubstances and these test substances further diluted 10 times, 100 timesand 1000 times with distilled water, their COX-2 inhibitory activitieswere measured by using the COX Inhibitor Screening Assay Kit (nontreatedgroup n=6, test substance group n=3). The enzyme activity value of eachtreated group was expressed as a relative % by the following formulabased on the COX-2 enzyme activity of the nontreated group which wasdefined as 100%. The results are shown in FIG. 9( a) and FIG. 9( b). InFIG. 9( a), the “Diclofenac Na” corresponds to the above-describeddiclofenac sodium aqueous solution, and the “Dic-C3-HA” to theabove-described aminopropanol-diclofenac-introduced sodium hyaluronateaqueous solution, and the “Dic-C2-HA” to the above-describedaminoethanol-diclofenac-introduced sodium hyaluronate aqueous solution.In FIG. 9( b), the “HA” corresponds to the above-described 200 μg/mlsodium hyaluronate aqueous solution.

Enzyme activity value (%)=(test substance-treated group)/nontreatedgroup×100

Consequently, the diclofenac-introduced hyaluronic acid derivatives(Dic-C3-HA and Dic-C2-HA) containing diclofenac, at a concentrationcorresponding to the concentration at which the diclofenac sodium singledrug showed obvious COX-2 inhibitory activity, did not show the COX-2inhibitory activity. The HA single drug also did not show the COX-2inhibitory activity. These results suggested that the effect of thediclofenac-introduced hyaluronic acid derivatives in vivo is not theaction of itself but may be referred from the diclofenac released fromHA.

As one of the reasons why the effect of the diclofenac-introducedhyaluronic acid derivatives in vivo (Examples 46 and 47) is superior tothat of the diclofenac single drug, it is considered that HA carries afurther larger amount of diclofenac to the COX-2 in the target cell dueto its affinity for the cell.

Example 49 Effects of Diclofenac-Introduced Hyaluronic Acid Derivativeon the Adjuvant-Induced Arthritis (AIA) Model in Rats 1) AdjuvantInduction

After heating 6 mg/ml Mycobacterium butyricum (Lot No. 2115687, Difco)at 121° C. for 20 minutes in an autoclave, it was injected into thefootpad of the right hind paw at a dose of 50 μl/joint, with a 29Gneedle-tipped syringe for insulin use (Terumo).

2) Administration of Test Substances

Test Substances:

(i) PBS solution of the 1% aminopropanol-diclofenac-introduced sodium(DS 18%) hyaluronate obtained in Example 19 (Diclofenac-HA)

(ii) PBS

As in the case of the adjuvant injection, the above-described testsubstances were administered under no anesthesia into the tibio-tarsaljoint cavities of both paws at a dose of 50 μl/joint with a 29Gneedle-tipped syringe for insulin use (Terumo), immediately after theadjuvant injection and on weeks 1, 2 and 3 after the injection (4 timesin total) (n=14).

4) Evaluation

Evaluation was performed prior to injection of the adjuvant and on days3, 5, 7, 10, 12, 14, 21 and 28 after the injection.

-   -   Body weight    -   Volumes of both hind paws (an equipment for measuring the volume        of hind-paw edema in rats and mice (TK-101CMP) manufactured by        Unicorn)

5) Results

Swelling ratio of the adjuvant-injected paws and non-injected paws wasmeasured by the following formula. Results of the swelling ratio of theadjuvant-injected paws are shown in FIG. 10( a), and results of theswelling ratio of the adjuvant-non-injected paws in FIG. 10( b). In FIG.10, the “Diclofenac-HA” means the above-described (i) PBS solution of 1%aminopropanol-diclofenac-introduced sodium (DS 18%) hyaluronate, and the“normal” means adjuvant- and test substance-non-administered groups. InFIG. 10, Δ indicates that there is a significant difference against PBSwith a level of significance of 0.05<p<0.1, and * indicates that thereis a significant difference against PBS with a level of significance of0.01<p<0.05, and ** indicates that there is a significant differenceagainst PBS with a level of significance of p<0.01.

${{Swelling}\mspace{14mu} {ratio}\mspace{14mu} (\%)} = {\frac{\begin{pmatrix}{{{paw}\mspace{14mu} {volume}\mspace{14mu} {on}\mspace{14mu} {the}\mspace{14mu} {measured}\mspace{14mu} {day}} -} \\{{paw}\mspace{14mu} {volume}\mspace{14mu} {prior}\mspace{14mu} {to}\mspace{14mu} {the}\mspace{14mu} {adjuvant}\mspace{14mu} {induction}}\end{pmatrix}}{{{volume}\mspace{14mu} {prior}\mspace{14mu} {to}\mspace{14mu} {the}\mspace{14mu} {adjuvant}\mspace{14mu} {induction}}\mspace{14mu}} \times 100}$

Edema was observed on the injected paw (R) from Day 3 after the adjuvantinduction, and the Diclofenac-HA showed the effect to suppress the edemavolume on Day 3, Day 5 and Day 21 postinduction, statisticallysignificantly in comparison with the PBS group, and also on Day 7, Day26 and Day 28 although not significant. Also at other time points, theDiclofenac-HA group showed low values at each time point although notsignificant.

An obvious swelling (secondary inflammation) was observed on thenon-injected paw (L) from Day 14 postinduction. Diclofenac-HAstatistically significantly suppressed this edema on Day 14 and Day 26postinduction. Furthermore, it showed tendency to suppress the edema onDay 21 and Day 28 although not significant.

In addition, since the rat adjuvant-induced arthritis (AIA) model isgenerally used as a model of rheumatoid arthritis which is arthritiscaused by an autoimmune disease, it is speculated that the substance ofthe present invention exert the effect on rheumatoid arthritis.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese patent application No. 2004-2478filed on Jan. 7, 2004 the entire contents of which are incorporatedhereinto by reference. All references cited herein are incorporated intheir entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, there are provided NSAIDs-introducedhyaluronic acid derivatives in which NSAIDs are bound to hyaluronic acidthrough a covalent bond via a spacer having a biodegradable region,DMARD-introduced hyaluronic acid derivatives in which DMARD is boundthereto through a covalent bond in the same manner, and pharmaceuticalagents which comprise these derivatives as an active ingredient. Sincethe NSAIDs-introduced hyaluronic acid derivatives and DMARD-introducedhyaluronic acid derivatives are sufficiently dissolved in buffers whichare used as the solvents of injection products and the like, they can beused as injection products which can be administered directly to theaffected site. Furthermore, the pharmaceutical agent of the presentinvention can be used for the treatment of arthritis, suppression ofinflammation and suppression of pain, and its parenteral administrationor topical administration (e.g., intra-articular administration) asfillers is also possible.

1. A hyaluronic acid compound in which a non-steroidal anti-inflammatorydrug is bound to hyaluronic acid through a covalent bond, wherein apartial structure of hyaluronic acid disaccharide unit into which theanti-inflammatory drug is introduced is represented by the followingformula (1):Y—CO—NH—R¹—(O—R²)_(n)  (1) wherein Y—CO— represents the glucuronic acidresidue of the hyaluronic acid disaccharide unit; R² represents ahydrogen atom or a non-steroidal anti-inflammatory drug residue, and atleast one R² is a non-steroidal anti-inflammatory drug residue;—NH—R¹—(O—)_(n), represents a spacer residue in a spacer compoundrepresented by H₂N—R¹—(OH)_(n) having n numbers of a hydroxyl group; R¹represents a linear or branched hydrocarbon group having from 2 to 12carbon atoms which may have a substituent; —CO—NH— represents an amidebond of a carboxyl group in the glucuronic acid as a constitutingsaccharide of the hyaluronic acid with an amino group in the spacercompound; wherein a hydroxyl group in the spacer compound forms an esterbond with a carboxyl group in the non-steroidal anti-inflammatory drugresidue; and n is an integer of from 1 to 3, and wherein a carbonylgroup in a hyaluronic acid residue constituting the hyaluronic acidcompound is present as an amide bond participating in the binding withthe spacer-binding anti-inflammatory drug residue or as a free carboxylgroup not participating therein.
 2. The hyaluronic acid compoundaccording to claim 1, wherein non-steroidal anti-inflammatory drug isselected from the group consisting of ketoprofen, naproxen, ibuprofen,flurbiprofen, acetylsalicylic acid, felbinac, fenbufen, mefenamic acid,diclofenac and etodolac.
 3. The hyaluronic acid compound according toclaim 1, wherein the non-steroidal anti-inflammatory drug is a compoundrepresented by the following formula (2):

wherein, R³ represents a substituent selected from the group consistingof a lower alkyl group, a lower alkoxyl group and a hydrogen atom; R⁴,R⁵ and R⁶ each independently represents a substituent selected from thegroup consisting of lower alkyl group, a lower alkoxyl group, a hydroxylgroup, a halogen atom, and a hydrogen atom; and each X is the same ordifferent and each represents a substituent selected from the groupconsisting of a lower alkyl group, a trifluoromethyl group, and ahalogen atom, wherein at least one of X is a halogen atom.
 4. Thehyaluronic acid compound according to claim 3, wherein the non-steroidalanti-inflammatory drug is a compound represented by the followingformula (7):

wherein R⁸ represents a substituent selected from the group consistingof a lower alkyl group, a lower alkoxyl group, and a hydrogen atom; andX¹ and X² each independently represents a substituent selected from thegroup consisting of a lower alkyl groups, a trifluoromethyl group, and ahalogen atom, wherein at least one of X¹ and X² represents a halogenatom.
 5. The hyaluronic acid compound according to claim 1, wherein thenon-steroidal anti-inflammatory drug is represented by the followingformula:


6. The hyaluronic acid compound according to claim 1, wherein thehyaluronic acid has a weight average molecular weight of from 500,000 to3,000,000.
 7. The hyaluronic acid compound according to claim 1, whereinthe hyaluronic acid compound has a degree of substitution of thenon-steroidal anti-inflammatory drug of from 0.1 to 80 mol % perrepeating disaccharide unit of hyaluronic acid.
 8. The hyaluronic acidcompound according to claim 1, wherein R¹ in formula (1) is selectedfrom the group consisting of an ethylene group, a trimethylene group anda propylene group, which may have one or more substituents.
 9. Thehyaluronic acid compound according to claim 1, which is obtainable by amethod comprising reacting hyaluronic acid with a spacer-boundnon-steroidal anti-inflammatory drug, or reacting a spacer-boundhyaluronic acid with a non-steroidal anti-inflammatory drug, andadjusting the reaction solution to alkaline conditions.
 10. Thehyaluronic acid compound according to claim 1, wherein a solutionobtained by dissolving the hyaluronic acid compound in an aqueous mediumto a concentration of 1.0% by weight is capable of passing through aporous filter having a pore size of 0.45 μm and a diameter of 25 mm, ata ratio of 2 mL per minute or more at a temperature of 24° C. under apressure of 5.0 kg/cm².
 11. The hyaluronic acid compound according toclaim 1, wherein a solution obtained by dissolving the hyaluronic acidcompound in an aqueous medium to a concentration of 1.0% by weight iscapable of passing through a porous filter having a pore size of 0.22 μmand a diameter of 25 mm, at a ratio of 2 mL per minute or more at atemperature of 24° C. under a pressure of 5.0 kg/cm².
 12. A hyaluronicacid compound solution which is capable of being pushed out from aninjector and which comprises the hyaluronic acid compound according toany one of claims 1 to 11 dissolved in an aqueous medium.
 13. Thehyaluronic acid compound solution according to claim 12, wherein theaqueous medium is an aqueous medium selected from the group consistingof phosphate buffered saline, saline and water for injection.
 14. Thehyaluronic acid compound solution according to claim 12, which issterilized through a filter.
 15. A pharmaceutical composition whichcomprises the hyaluronic acid compound according to claim 1 as an activeingredient and a pharmaceutically acceptable carrier.
 16. Thepharmaceutical composition according to claim 15, which is an arthritistreating agent, an anti-inflammatory medicament or an analgesic.
 17. Thepharmaceutical composition according to claim 15, which is useful forparenteral administration.
 18. The pharmaceutical composition accordingto claim 17, which is an injection useful for topical administration.19. The pharmaceutical composition according to claim 17, which is aninjection useful for intra-articular administration.
 20. Apharmaceutical composition which is capable of being pushed out from aninjector and which comprises a solution in which the hyaluronic acidcompound according to claim 1, as an active ingredient, is dissolved inan aqueous medium.
 21. A kit for injection of a hyaluronic acidcompound, which comprises the hyaluronic acid compound solutionaccording to claim 12 which is filled in an injector capable of pushingout the solution.
 22. The kit according to claim 21, wherein the filledsolution is a pharmaceutical composition which comprises a hyaluronicacid compound as an active ingredient and a pharmaceutically acceptablecarrier, wherein the hyaluronic acid compound is a hyaluronic acidcompound in which a non-steroidal anti-inflammatory drug is bound tohyaluronic acid through a covalent bond, wherein a partial structure ofhyaluronic acid disaccharide unit into which the anti-inflammatory drugis introduced is represented by the following formula (1):Y—CO—NH—R¹—(O—R²)_(n)  (1) wherein Y—CO— represents the glucuronic acidresidue of the hyaluronic acid disaccharide unit; R² represents ahydrogen atom or a non-steroidal anti-inflammatory drug residue, and atleast one R² is a nonsteroidal anti-inflammatory drug residue;—NH—R¹—(O—)_(n) represents a spacer residue in a spacer compoundrepresented by H₂N—R¹—(OH)_(n) having n numbers of a hydroxyl group; R¹represents a linear or branched hydrocarbon group having from 2 to 12carbon atoms which may have a substituent; —CO—NH— represents an amidebond of a carboxyl group in glucuronic acid as a constituting saccharideof the hyaluronic acid with an amino group in the spacer compound;wherein a hydroxyl group in the spacer compound forms an ester bond witha carboxyl group in the non-steroidal anti-inflammatory drug residue;and n is an integer of from 1 to 3; and wherein a carbonyl group in ahyaluronic acid residue constituting the hyaluronic acid compound ispresent as an amide bond participating in the binding with thespacer-binding anti-inflammatory drug residue or as a free carboxylgroup not participating therein.
 23. A medical injection kit which issealed with a plunger for medicament extrusion in such a manner that itcan be slid and which comprises a syringe filled with a solution inwhich the hyaluronic acid compound according to claim 1 is dissolved inpharmaceutically acceptable phosphate buffered saline, saline or waterfor injection.